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From the * Program for the Topical Prevention of
Conception and Disease (TOPCAD) and the
Department of Obstetrics and Gynecology, Rush
University Medical Center, Chicago, Illinois; and the
Department of Medicinal Chemistry and
Pharmacognosy, University of Illinois at Chicago, Health Sciences Center,
Chicago, Illinois.
Present address: Thermo Fisher Scientific,
2555 Kerper Blvd, Dubuque, IA 52002.
|| Present address: Chembiotek, International
Biotech Park, Bio Research Centre, Mulshi, Hinjewadi, Pune 411057, India.
| Correspondence to: Dr Robert A. Anderson, Jr, Ob/Gyn Research, Rush Medical Center, Chicago, IL 60612 (e-mail: robertan{at}corecomm.net). |
| Received for publication April 3, 2008; accepted for publication October 7, 2008. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Key words: Signal transduction, mechanism, nitric oxide synthase, cGMP, guanylate cyclase, protein kinase G, protein kinase A, protein tyrosine kinase
Unplanned pregnancies and abortion procedures are another risk factor to women's reproductive health. Nearly half of unplanned pregnancies in the United States are terminated (Henshaw, 1998), at a cost of more than $5 billion (Trussell, 2007). Improved methods for contraception and prevention of sexually transmitted disease will help to reduce population growth and demand for natural resources, and will improve the general health of both genders.
Microbicidal products (microbicides), with or without contraceptive properties, are being developed (D'Cruz and Uckun, 2004). These products are intended for prophylactic topical use. In 2004, about 60 candidates had entered various phases of development (Mantell et al, 2005). Recent late-stage failures (European AIDS Treatment Group, 2006; Alliance for Microbicide Development, 2007; BBC News, 2007; Lirri, 2007) of several microbicides and one vaccine underscore the importance of continued discovery.
Sulfuric acid–modified mandelic acid (SAMMA) is a carboxylated oligomer with a molecular weight of 1.5 to 1.9 kDa. It is contraceptive, with activity against several pathogens, including among others, HIV, HSV, N gonorrhoeae, and C trachomatis (Zaneveld et al, 2002).
SAMMA is an acronym that describes several possible products formed from reaction of D,L-mandelic acid with sulfuric acid. The exact nature of the reaction (hence, the product) depends on reaction conditions. Among possible reaction products are sulf(on)ated ring structures (Roberts and Caserio, 1964b), ethers (Roberts and Caserio, 1964a), cyclic dimers, and acid-catalyzed polyesters (Whitesell and Pojman, 1990). A proposed ether product with activity against HIV-1 and a polyester (with weak activity against HIV-1) have been described (Ward et al, 2008). The product described herein, formed under proprietary reaction conditions, and distinct from those mentioned above, will be referred to as PPCM, a code name assigned by Yaso Biotechnology Inc, the licensee for this material (Zaneveld et al, 1999). The form of SAMMA used to describe its microbicidal properties (Zaneveld et al, 2002; Cheshenko et al, 2004; Chang et al, 2007) and effects on spermatozoa/conception (Zaneveld et al, 2002; Anderson et al, 2006) is PPCM, a carboxylated oligomer. Structural characterization of this compound will be considered in a separate document (Krunic et al, unpublished).
PPCM commercialization and design of second generation products based on the PPCM prototype require understanding of its mechanisms of action. PPCM exerts its antiviral activity in part by preventing binding and entry of viruses to their target cells (Cheshenko et al, 2004). Its prevention of HIV transmission by dendritic cells (Chang et al, 2007) suggests other mechanisms of antiviral activity as well. Mechanisms for its antibacterial activity are unknown.
Part of PPCM's contraceptive activity may be attributable to its induction of premature acrosomal loss (AL; Zaneveld et al, 2002). This process is Ca2+-dependent, with Ca2+ entry into the spermatozoa mediated by T-type (Cav 3.x) voltage-dependent Ca2+ channels. This is distinct from the acrosome reaction induced by zona pellucida or progesterone (Anderson et al, 2006).
This study was carried out to extend our understanding of how PPCM induces premature AL by examining signal transduction downstream from Ca2+ entry. In view of the relatively low percentage of human spermatozoa that respond to stimuli of AL (typically, 30% to 35%; eg, Anderson et al, 1992; Revelli et al, 1999; Liu et al, 2008), a pharmacologic approach was taken, considering only spermatozoa with potential for AL in vitro as the dependent variable. Other approaches such as measurement of transduction pathway intermediates or enzymatic studies are less informative in this instance, because results would include activities derived from the bulk of spermatozoa that are nonresponsive, at least in vitro.
The results suggest that PPCM-induced AL (PAL) occurs by a nitric oxide (NO)–dependent process, through activation of endothelial NO synthase (eNOS), and downstream activation of soluble guanylate cyclase and type II cyclic guanosine monophosphate (cGMP)-dependent protein kinase (protein kinase G; cGK-II). NO donors also induce AL. However, signal transduction elements responsible for NO donor-induced AL and PAL are independent. The data also point to the importance of activity of the NO/cGMP/cGK pathway as a determinant of acrosomal status.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Synthesis of PPCM and Derivatives
PPCM is an oligomer with average molecular weight 1550 to 1860. It is
synthesized by a proprietary method by reacting D,L-mandelic acid
with sulfuric acid. The sodium salt is prepared by reacting the free acid with
alcoholic NaOH, yielding an off-white powder.
The NO donor 3-nitrooxypropan-1-ol (nitrooxypropanol), a nitrate semiester, is prepared by reacting silver nitrate with 3-bromo-1-propanol in acetonitrile. Silver nitrate (18.7 g, 110 mmol) was dissolved in acetonitrile (200 mL). 3-Bromo-1-propanol (18.9 g; 100 mmol), dissolved in 25 mL of acetonitrile, was added and protected from light. The reaction mixture was stirred at room temperature for about 96 hours; after 2 hours, a yellow precipitate (AgBr) was formed. The reaction mixture was filtered through a cellite pad to remove AgBr. The solvent was removed by rotary evaporation to yield a pale yellow to clear oil, which was dissolved in dichloromethane. This solution was extracted with saturated NaCl in water to remove residual silver as AgCl, and dried over anhydrous sodium sulfate. After solvent removal by rotary evaporation and distillation (50 mm Hg), nitrooxypropanol (colorless to pale yellow oil) was obtained in about 62% yield. Identity was confirmed with infrared (IR) spectroscopy and 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy. IR spectrum: 3359 cm–1 (OH); 2964 and 2896 cm–1 (CH); 1626 cm–1 (NO3 asymmetric); 1281 cm–1 (NO3 symmetric); 871 cm–1 (N=O); 759 cm–1 (NO2 bend). 13C NMR spectrum: 70.62 ppm (C1), 58.01 ppm (C2), 29.30 ppm (C3). 1H NMR spectrum (in CDCl3): 4.62 ppm (2H, H1), 3.78 ppm (2H, H3), 1.99 ppm (2H, H2), 1.70 ppm (1H, OH); numerical prefixes refer to number of protons, and suffixes refer to position on the structure. Nitrooxypropanol releases NO, measured by nitrite formation (Griess reaction). Nitrooxypropanol (0.1 mM) was reacted with 50 mM L-cysteine at 37°C for 24 hours. Maximum nitrite formation approaches 58.3 µM (data not shown).
A PPCM derivative (NO donor-substituted poly-acidic polymer [NOSPPA]) was prepared, in which nitrooxypropanol was substoichiometrically esterified (approximately 23% substitution) to PPCM carboxyl groups. PPCM (3.19 g; 23.8 acid mEq) was dissolved in 500 mL dimethyl formamide (DMF) in a flask with attached drying tube and cooled to 0°C (ice bath). Excess 1,1'-carbonyldiimidazole (9.73 g; 60 mmol; coupling agent) was dissolved in 40 mL DMF and added to the stirred PPCM solution. After further addition of 30 mL DMF, the reaction was continued for 45 minutes with stirring at 0°C. Nitrooxypropanol (0.86 g; 7.1 mmol) in 5 mL dry DMF was added drop-wise over a 2-minute period. The reaction temperature was allowed to rise to ambient temperature. The reaction was continued for 15 hours with stirring at ambient temperature. The mixture was decanted into 1000 mL water and acidified to pH 1.6 with 6 N HCl. The precipitate was suction-filtered, washed with water (two 200-mL portions), and suction-filtered. The precipitate was lyophilized to yield 2.78 g of NOSPPA-23 (approximately 73% yield). Elemental analysis (calculated and observed): C, 66.4% and 66.0%; H, 4.5% and 4.8%; N, 1.9% and 2.0%. IR spectrum: 1722 cm–1 (COO-R); 1712 cm–1 (COOH); 1629 cm–1 (asymmetric NO2 stretch); 1280 cm–1 (symmetric NO2 stretch). NOSPPA-23 releases NO, measured with the Griess reaction. NOSPPA-23 (0.1 mg/mL; approx 63 µM) was reacted with 50 mM L-cysteine at 37°C for 24 hours; nitrite formation approached 11.3 µM (data not shown).
Human Subjects
In each experiment, fresh semen was collected from 2 or 3 out of a total of
9 healthy donors (age [
± SEM]
= 32 ± 3.5 years). Details regarding medical histories and inclusion
criteria for these individuals have been presented
(Anderson et al, 2006). Donors
participated in this study with informed consent. The study was approved by
and in compliance with the medical center institutional review board. Semen
was of high quality, with average volume of 4.8 ± 0.68 mL and average
sperm count of 82 (90% confidence limits = 70.1–97.2) x
106 cells/mL. Initial fraction of motile sperm for all experiments
was 70.2% (66.90% to 73.42%).
Procedures
Preparation of Spermatozoa and Induction of AL—
Within the context of this study, AL refers to the disruption of the sperm
acrosome in response to a treatment or chemical entity. No inference is made
as to whether this response is identical to a physiological acrosome reaction,
during which the acrosome is also lost.
Within 90 minutes of collection, liquefied semen samples (2 or 3) were pooled after a cursory examination of sperm motility and count by light microscopy with a Neubauer hemacytometer (bright field, x400). The pooled sample was divided for each assay, such that approximately 0.1 mL (total sperm number = 5 x 106) was layered onto 1 mL of 11% (wt/vol) buffered Ficoll (containing 120 mM NaCl and 25 mM HEPES, pH 7.4), in plastic centrifuge tubes. Samples were centrifuged at 15 000 x g-min (22°C to 24°C), and the supernatant was aspirated from the sperm pellet. The pellet was resuspended in modified Biggers, Whitten, and Whittingham (BWW; Biggers et al, 1971) medium (less albumin), and the suspension was recentrifuged at 1000 x g-min (22°C to 24°C). The supernatant was aspirated, and the pellet was resuspended in 1 mL BWW medium. Details of sperm preparation have been described (Anderson et al, 1992, 1994).
After equilibration of washed spermatozoa for 10 minutes at 37°C, transduction pathway modulators were added, as indicated. After an additional 10 minutes at 37°C, AL was induced, either by addition of stimulus (PPCM, dbcAMP, hANP, or progesterone) or by adding CaCl2, as indicated. Fifteen minutes after AL induction, spermatozoa were fixed with buffered glutaraldehyde and stained with rose bengal and Bismarck brown for acrosome visualization (Anderson et al, 1992, 2006). Approximately 450 cells were scored per slide; typical cell counts in a given experiment ranged from approximately 400 to 700. From 3 to 4 replicates were measured in each experiment. The total number of replicates for each condition are presented in "Results." Data are expressed as averages, with 90% confidence limits, of percentage of AL induced by a maximally stimulating concentration of A23187 (Anderson et al, 1992). The percentage of spermatozoa lacking acrosomes after treatment with A23187 under these conditions is 30.7% (90% confidence limits = 30.5% to 31.0%; n = 30).
In some instances, stock solutions of modulators (ie, KT5720, KT5823, progesterone, genistein, ODQ) were prepared in dimethyl sulfoxide (DMSO). The DMSO concentration was not greater than 1.5% (vol/vol). Equivalent concentrations of DMSO were added to the appropriate controls. DMSO is without effect on sperm motility and acrosomal status under these conditions.
Estimation of NO Production— NO production by spermatozoa was estimated with a modification of the method of Revelli et al (1999). The method determines stimulus-induced production of nitrite, measured spectrophotometrically with the Griess reaction.
Approximately 30 x 106 spermatozoa were suspended in arginine-supplemented (2 mM) BWW medium. Incubations with different additions were carried out for 1 hour at 37°C, after which cells were disrupted with 0.2% Triton X-100, followed by 1 freeze-thaw cycle (–80°C to 37°C). Particulates were sedimented at 80 000 x g-min).
Equal volumes of supernatant and modified Griess reagent were combined and
reacted in the dark for 10 minutes before reading the absorbance at 540 nm.
Nitrite formation (pmol nitrite/106 cells) was quantified with a
nitrite standard linear curve (r2 = 0.9997), at 5
concentrations ranging from 0.25 to 5.0 µM, and is expressed as
± SEM.
Data Collection and Analysis
Frequency (%) data were subjected to arcsine transformation before analysis
(Sokal and Rohlf, 1981b).
Values are presented as average percentage of maximal AL, with 90% confidence
limits. Analysis of variance and the Newman-Keuls multiple range test were
used to identify differences among treatment groups within individual
experiments. Where appropriate, a 2-tailed unpaired t test was used
to determine the significance of differences between treated samples and their
respective controls. Differences among treatment groups were considered
significant at P < .05. Differences were not considered
significant at P > .1.
Determination of Independence of Actions of 2 AL Stimuli
In some instances, inhibitors of signal transduction pathways induced AL
when added alone. To determine the effect of these agents on stimulus-induced
AL, data were examined for interactive effects.
Interactive effects of 2 agents on a normally distributed variable can be evaluated with 2-way analysis of variance, looking for deviations from additive effects. However, this cannot be applied to frequency data, because of constraints placed on the possible range of values (0% to 100%). This is clearly seen if 2 agents, each of which produces 60% inhibition of some biological activity, are added in combination. The expected outcome is somewhat less than 100% inhibition, assuming that the 2 agents acted independently. In this instance the predicted effect would be 84% inhibition, unless interactive effects alter the outcome.
AL data are binary. With binary data, the probability of an event not occurring in the presence of 2 independent agents is equal to the product of the probabilities of that event not occurring in the presence of each agent alone. If agent A produces AL in 90% of the spermatozoa, and agent B causes the same outcome, but by an independent mechanism, the expected frequency of AL when both A and B are added in combination to the cells is (1 – [1 – 0.9][1 – 0.9]) x 100 = (1 – 0.01)(100) = 99%. A detailed discussion of this subject is available (Piegorsch and Margolin, 1989).
Interactive effects were evaluated by comparing the observed with the
predicted responses to combined treatment with inhibitor (or second stimulus)
and stimulus, based on the response to the 2 agents, each added alone. The
interactive effect was evaluated with the log likelihood ratio test (G test)
of independence (Sokal and Rohlf,
1981a). When the second stimulus was a pathway inhibitor, the
extent to which that agent inhibited the response to the primary stimulus was
estimated with the following equation:
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Results |
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Neither S-methylthiocitrulline, selective for the neuronal isoform, nNOS (NOS-1; Furfine et al, 1994), nor 1400W, selective for iNOS (Garvey et al, 1997), inhibits PAL (Figure 1).
PAL Requires Soluble Guanylate Cyclase
LY83583 (50 nM), an inhibitor of soluble and receptor-linked guanylate
cyclases (Anand-Srivastava and Trachte,
1993) inhibits PAL (Figure
2). Similar to the iNOS and eNOS inhibitors, LY83583 induces AL in
the presence of added Ca2+. LY83583 is without effect on AL when
Ca2+ is not added (2.2% [0.82% to 4.53%]). PAL is nearly 90%
inhibited by LY83538.
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ODQ (100 nM), a selective soluble guanylate cyclase inhibitor (Garthwaite et al, 1995), inhibits PAL, but not hANP-induced (t = 0.54, df = 6, P > .1) AL (Figure 2). In the presence of added Ca2+, ODQ alone exerts a small stimulatory effect on AL. ODQ has no effect on AL in the absence of added Ca2+ (0.5% [–0.27% to 3.70%]).
When reactions are carried out with added Ca2+, responses to 0.25 µg/mL PPCM and 0.1 nM hANP are 50% (49.0% to 51.4%; n = 18) and 59% (56.4% to 61.3%; n = 4) maximal AL, respectively. The predicted response to combined addition of these agents is 79.5%, if they act independently. The observed response to PPCM and hANP added in combination is 79% (76.7% to 81.7%; n = 4).
PAL Is Antagonized by cGK Inhibition
The selective cGK inhibitor KT5823
(Nakanishi, 1989) completely
inhibits PAL. The AL response to combined addition of 2 µM KT5823 and 0.25
µg/mL PPCM represents a highly antagonistic interaction
(Table 1).
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PPCM Promotes NO Formation
PPCM increases sperm production of NO, as measured by an increase in
nitrite formation. Nitrite formation is increased by 57% as compared with
control reactions containing no additions
(Table 2). This response is
inhibited by approximately 88% by 10 µM
NG-nitro-L-arginine.
NG-nitro-L-arginine reduces baseline nitrite by
approximately 22%; variability of the data precludes a significant difference
(Table 2). NO production in
response to PPCM is much lower than that in response to the NO donor SNAP,
although both agents produce comparable (50% to 80%) AL.
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AL by PPCM and NO Donors Occurs by Different Pathways
Unlike PAL (Anderson et al,
2006), SNAL does not require added Ca2+. SNAP (0.4 mM)
induces, in the presence and absence of added Ca2+, 81% (79.6% to
83.1%; n = 8) and 79% (76.9% to 80.4%; n = 16) maximal loss, respectively
(t = 1.70, P > .1).
As expected, SNAL is sensitive to the soluble guanylate cyclase inhibitor ODQ. However, inhibition is incomplete (approximately 50%) at 100 nM ODQ (Figure 3), a concentration sufficient to completely block PAL (Figure 2). Similar incomplete inhibition by ODQ occurs in AL induced by the nitrate ester NO donor nitrooxypropanol (63% inhibition at 100 nM ODQ). Even at 10-fold higher concentration, inhibition of SNAL approaches only 80% (Figure 4).
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Unexpectedly, SNAL is unaffected by the cGK inhibitor KT5823 when added at a concentration (2 µM) that completely inhibits PAL. Apparent lack of cGK involvement is also demonstrated for several other NO donors, independent of mechanism of NO release (Table 3).
Effects of several other cGK inhibitors on PAL and SNAL were examined. PAL
is inhibited by KT5823 (selective for cGK-I and cGK-II;
Mishra et al, 2001;
Tischkau et al, 2004) and by
Rp-8-pCPT-cGMPS (inhibits cGK-I and cGK-II;
Butt et al, 1994;
Gambaryan et al, 1996; also
inhibits protein kinase A (PKA) at concentrations effective against cGK;
Smolenski et al, 1998).
However, PAL is unaffected by either Rp-8-Br-PET-cGMPS (inhibits cGK-I
and cGK-Iβ; Schwede et al,
2000; also inhibits cGMP-specific phosphodiesterase type 5
[PDE-5]; Butt et al, 1995) or
DT-3 (selective for cGK-I; Peluso
and Pappalardo, 2004). Of the inhibitors examined, only
Rp-8-pCPT-cGMPS weakly inhibits SNAL (Table
1).
The cGK inhibitors, KT5823 and Rp-8-pCPT-cGMPS (nonselective for cKG
isotype), induce AL when added alone in the presence of added Ca2+.
Under similar conditions, somewhat lower (approximately 21% maximal loss) AL
is induced by the cGK-I inhibitor Rp-8-Br-PET-cGMPS; however, DT-3, selective
for cGK-I, is without effect (Table
1). All inhibitors are without effect on AL by themselves in the
absence of added Ca2+.
The ability of cGK inhibitors to induce Ca2+-dependent AL is similar to that seen for NOS and guanylate cyclase inhibitors (Figures 1 and 2), suggesting that the resting or basal state NO/cGMP/cGK transduction pathway may be a determinant of acrosomal status, possibly through control of Ca2+ entry. AL induced by NG-nitro-L-arginine (inhibits NOS), LY83583 (inhibits guanylate cyclase), and KT5823 (inhibits cGK) are inhibited by the Ca2+ channel blocker nifedipine (Table 4). Nifedipine alone has a negligible effect on AL.
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The PKA inhibitor KT5720 (Nakanishi, 1989) inhibits SNAL, but similarly to inhibition by ODQ, inhibition is incomplete. The concentration of KT5720 used is sufficient to completely block AL induced by dbcAMP (Figure 5). KT5720 is ineffective against PAL. SNAL was examined in the presence of KT5720 and ODQ, added alone or in combination, to determine possible interaction between the cAMP and cGMP pathways. There is no interaction between these inhibitors (Figure 5).
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PPCM at 0.25 µg/mL and 0.25 mM SNAP induce 54% (50.6% to 58.7%) and 56% (54.4% to 58.8%) maximal AL, respectively. Observed response to combined addition of PPCM and SNAP is 82% (79.4% to 84.5%), reflecting no interactive effect of PPCM and SNAP (G test of independence = 0.345, df = 1, P > .1).
The above results suggest that AL mediated by NO produced by 2 different types of stimuli (PPCM and NO donors) occurs by independent mechanisms. Covalent attachment of an NO donor to PPCM may result in a derivative with enhanced biological activity.
To test this hypothesis, the NO donor nitrooxypropanol was covalently coupled to PPCM (see "Materials and Methods") to form a derivative in which approximately 23% of available carboxyl groups are substituted with the NO donor by esterification (NOSPPA-23).
NOSPPA-23 induces AL in the absence of extracellular Ca2+ (Figure 7). The response to 0.02 µg/mL NOSPPA-23 in the absence of added Ca2+ (47%) is nearly 7-fold higher than the predicted response (6.8%) to an equivalent amount of the NO donor (36 nM nitrooxypropanol) from which NOSPPA-23 was derived (based on dose-response of nitrooxypropanol-induced AL; Figure 8, panel B). AL induced by nitrooxypropanol is independent of added Ca2+ (data not shown); PPCM is without effect in the absence of added Ca2+ (Anderson et al, 2006).
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The response to the same concentration of NOSPPA-23 in the presence of added Ca2+ is over 8-fold greater than that predicted (10.9%) from combined addition of the equivalent concentrations of PPCM (19 ng/mL) and nitrooxypropanol, assuming independent mechanisms of action (Figure 7). The increase in AL in the presence of NOSPPA-23 over that seen with the equivalent concentration of NO donor alone is over 21-fold higher than the predicted increase over the NO donor–induced AL because of the addition of PPCM. The ED50 of NOSPPA-23 in the presence of Ca2+ (8 ng/mL, or approximately 4.8 nM; Figure 8, panel A) is more than 30 times less than the ED50 for PPCM, on the basis of mass. On a molar basis, NOSPPA-23 is approximately 35-fold more active than PPCM. The ED50 of NOSPPA-23 in the absence of added Ca2+ (27.9 ng/mL) is equivalent to 40 nM NO donor, based on the nitrogen content (2.0%) of this material. This is approximately 3-fold less than the ED50 of the parent NO donor, nitrooxypropanol (120 nM; Figure 8, panel B).
In no instance is average sperm motility reduced more than 5% (after
addition of either 50 nM LY83583 or 0.5 µM
S-methyl-L-thiocitrulline) from control values (no additions) as a
result of treatment. Motility after addition of transduction pathway
inhibitors ranges from 95% to 108% of control. Average motility loss caused by
0.25 µg/mL PPCM (concentration required to produce 50% maximal AL) is 0.1%
(–0.8% to 1%). The fraction of motile spermatozoa increases in response
to either 20 µM dbcAMP or 16 nM progesterone (by 24% and 35%, respectively;
P < .05). No relation exists between AL and the change in
percentage of motile spermatozoa (Kendall's rank order correlation coefficient
= –0.156, n = 10, P > .1). The likelihood of changes
in AL being secondary to decreased cell viability is therefore minimal.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Our examination of possible involvement of signal transduction pathways in PAL was based in part on putative mechanism(s) of the mammalian acrosome reaction. Reviews on the subject have considered several post-Ca2+ entry pathways and second messengers. These include, among others, increased protein tyrosine phosphorylation, increased cAMP levels, activation of PKA, increased adenylate cyclase activity and activation of L-type voltage-dependent calcium channels (Breitbart and Spungin, 1997; Benoff, 1998; Guraya, 2000). cGMP (Anderson et al, 1995; Kobori et al, 2000) and NO (Kameshwari et al, 2003) may also participate. Our objective was to determine whether PAL is mediated by pathways common to those mediating the physiological acrosome reaction.
eNOS Is a Transduction Element Leading to PAL
PAL requires NOS activity. Based on the activities of several NOS
inhibitors (Figure 1), we
conclude that PAL is mediated by NO produced by eNOS, or an eNOS-like isoform
of NOS.
NO is produced by different isoforms of NOS (Knowles and Moncada, 1994). eNOS and nNOS have been identified in spermatozoa by immunohistochemistry (O'Bryan et al, 1998) and Western blotting (Revelli et al, 1999). iNOS may also be present in spermatozoa (Balercia et al, 2004). Involvement of eNOS in PAL is consistent with its localization to the postacrosomal region and equatorial segment (O'Bryan et al, 1998).
The nonselective NOS inhibitor, NG-nitro-L-arginine, is the most effective at inducing AL when added alone in the presence of added Ca2+. No effect is observed when Ca2+ is not added (data not shown). The nNOS inhibitor S-methylthiocitrulline is without effect on PAL or on AL when added alone, either with or without added Ca2+ (Figure 1), suggesting that nNOS does not contribute substantially either to PAL or to resting NO levels.
Soluble Guanylate Cyclase Is a Transduction Element Leading to PAL
PAL is inhibited by guanylate cyclase inhibitors LY83583 and ODQ
(Figure 2). LY83583 inhibits
soluble and particulate (receptor-linked) guanylate cyclases
(Mulsch et al, 1988) and
inhibits hANP-induced AL (Anderson et al,
1994). hANP exerts its effect through the particulate enzyme
(Anand-Srivastava and Trachte,
1993). The soluble guanylate cyclase inhibitor ODQ
(Garthwaite et al, 1995) has
no effect on hANP-induced AL (Figure
2). From the inhibitor data, PPCM could be acting through
particulate, as well as soluble, guanylate cyclase. Particulate enzyme
involvement would be supported by antagonism between PPCM and hANP as AL
stimuli. However, hANP and PPCM induce AL by independent mechanisms
("Results, PAL Requires Soluble Guanylate Cyclase").
Therefore, PAL is likely mediated by activation of only soluble guanylate
cyclase.
cGK-II Is a Transduction Element Leading to PAL
There are 2 isotypes of cGK, cGK-I and cGK-II. cGK-I exists in 2 isoforms
(cGKI- and -β; Smolenski et
al, 1998). Failure of inhibitors that are selective for isoforms
of cGK-I to inhibit PAL (Table
1) argues against involvement of the I isotype. Spermatozoa from
cGK-I–deficient knockout mice are fertile and can undergo
"spontaneous" acrosome reaction
(Hedlund et al, 2000),
suggesting that cGK-I plays little, if any, role in this aspect of sperm
function. AL is induced by NO that is likely produced/released at or near the
plasma membrane in response to PPCM. This favors involvement of the
membrane-bound (Ruth, 1999;
Hofmann et al, 2004) cGK-II
for NO production responsible for PAL.
Resting Level of cGMP as a Determinant of Acrosomal Status
This study extends our early work on induction of AL by agents that lower
resting cGMP levels. LY83583, by itself, promotes AL in a
Ca2+-dependent manner (Anderson
et al, 1995). Our observation with LY83583 has been extended to
include induction of AL by compounds that inhibit different elements of the
NO/cGMP/cGK pathway. Induction of Ca2+-dependent AL by these agents
is inhibited by the L-type (Cav 1.x) Ca2+
channel blocker nifedipine (Bean,
1989; Table 4).
These findings support our earlier contention that basal cGMP levels exert an
inhibitory effect on calcium channel activity in spermatozoa, as they do in
other tissue (Keef et al,
2001), and exert control over acrosomal status.
Interestingly, the cGK-I inhibitor cGMPSRp-8-Br-PET-cGMPS, added alone, is a stimulus of AL in the presence of Ca2+. Reasons for this effect are not clear. Our data with other cGK-I inhibitors, and immunologic work by others (Willipinski-Stapelfeldt et al, 2004), suggest that cGK-I is not active in human spermatozoa. cGMPSRp-8-Br-PET-cGMPS is also an inhibitor of cGMP-specific PDE-5 (Butt et al, 1995). AL induced by this inhibitor may be caused by an increase in cGMP, secondary to inhibition of PDE-5.
Biphasic activity of cGMP such as seen in this study (ie, induction of AL by either an increase or a decrease in cGMP) has been observed in other cells. Elevated cGMP directly activates Ca2+ channels (Kaupp, 1991), but inhibits Ca2+ channels via cKG activation (Hofmann et al, 1992).
Importance of NO in Spermatozoal Function
NO exerts several effects on spermatozoa, including induction of acrosome
reaction (Kameshwari et al,
2003), increased cGMP (Revelli
et al, 2001) and cAMP (Herrero
et al, 2000), and increased protein tyrosine phosphorylation
(Kameshwari et al, 2003).
Excessive NO production may cause premature AL, leading to infertility
(Herrero and Gagnon, 2001).
However, spermatozoa have resting levels of NO that may contribute to cGMP
levels in noncapacitated spermatozoa
(Revelli et al, 2002) and thus
preserve an intact acrosome.
PAL and AL Induced by NO Donors Occur by Different Pathways
PAL, although mediated by NO, differs from AL induced by NO donors (eg,
SNAP). AL induced by SNAP or other NO donors is not inhibited by cGK
inhibitors (Tables 1 and
3), regardless of the mechanism
of NO release. SIN-1 releases NO spontaneously by an oxygen-dependent
mechanism (Feelisch et al,
1989); 4-phenyl-3-furoxancarbonitrile release of NO is
thiol-activated and NO release by nitroprusside depends on redox activation
(Ferioli et al, 1995); release
of NO from SNAP is spontaneous (Noack and
Feelisch, 1991), but is also dependent on other factors, including
cell membrane components (Kowaluk and
Fung, 1990).
PAL, but not SNAL, depends on added Ca2+ ("Results;" Anderson et al, 2006). This difference is not unexpected, assuming that Ca2+ entry promoted by PPCM occurs upstream from NO formation. SNAL, but not PAL, is sensitive to PKA inhibition by KT5720 (Table 5, Figure 5). Both PAL and SNAL are inhibited by ODQ (Figure 2; Table 5), strongly suggesting that cGMP mediates AL induced by both agents.
The most common effectors of cGMP are 1) type 3 phosphodiesterase (PDE-3) inhibition, resulting in increased cAMP levels/PKA activation (Marletta, 2003); 2) cGK activation; and 3) opening of cyclic nucleotide-gated cation channels (Felix, 2005), through which Ca2+ can pass. None of these possibilities are completely consistent with the available data regarding SNAL.
First, inhibition of SNAL by ODQ and inhibition of SNAL by KT5720 appear to be independent (Figure 5). If cGMP were acting through PKA activation (secondary to increased cAMP levels or to crossover activation of PKA by cGMP; Keef et al, 2001), then the effect of PKA inhibition would be influenced by the extent to which cGMP was produced (affected by guanylate cyclase inhibitors, such as ODQ). An interactive effect between ODQ and KT5720 should exist if PKA were being activated by cGMP under these conditions. This does not occur.
Second, SNAL is essentially unaffected by cKG inhibitors; it is unaffected by the cGK inhibitor KT5823 and only approximately 20% inhibited by Rp-8-pCPT-cGMPS. However, Rp-8-pCPT-cGMPS also inhibits PKA at concentrations that inhibit cGK (Smolenski et al, 1998). Inhibition by this inhibitor is similar to inhibition by the PKA inhibitor KT5720 (24%; Table 1; Figure 5), suggesting that its effect may be attributable to PKA inhibition.
Third, SNAL does not require addition of Ca2+. This minimizes the possibility that SNAP is acting through gated Ca2+ channels. Based on inhibition by ODQ, approximately 80% of AL induced by SNAP (and possibly other NO donors) is dependent on cGMP, which likely acts on an effector other than cGK, gated Ca2+ channels, or PDE-3.
PKA Is a Transduction Element Leading to SNAL
The inhibitor response profile of SNAL is similar to that observed in other
tissues treated with NO donors. Vila-Petroff et al
(1999) reported an increase in
adenylate cyclase activity in myocytes in response to SNAP, and showed that
inhibition of SNAP-induced contractile response by ODQ and the PKA inhibitor
Rp-8CPT-cAMPS are independent, thus separating increased cGMP levels from PKA
activation. A direct effect of NO on adenylate cyclase is suggested.
Results from combined addition of KT5720 and ODQ (Figure 5) suggest that these agents inhibit SNAL independently. This finding, together with less than complete inhibition of SNAL by ODQ, is consistent with other work suggesting that NO may have a direct effect on adenylate cyclase independent of increased cGMP (Vila-Petroff et al, 1999; Herrero and Gagnon, 2001).
KT5720 inhibits SNAL by only approximately 24% at a concentration that is nearly 20-fold higher than its Ki (Kase et al, 1987) for PKA (a concentration that remains selective for PKA; see Nakanishi, 1989). This concentration completely inhibits AL induced by dbcAMP (Figure 5). Higher inhibition of SNAL would be expected if all activity caused by cGMP were mediated by PKA activation. Similarly, not all SNAL can be explained by changes in cGMP. ODQ inhibits SNAL by only 80% at a concentration approximately 10-fold greater than its Ki for guanylate cyclase. NO and cGMP produced in the same cell may exert actions through activation of separate transduction pathways (Hofmann et al, 2006).
Protein Tyrosine Phosphorylation Is a Transduction Element Leading to SNAL but not to PAL
Tyrosine phosphorylation is increased by NO donors
(Herrero and Gagnon, 2001).
This is consistent with our observation that genistein, a PTK inhibitor
(Hidaka and Kobayashi, 1992),
inhibits SNAL. However, PAL is unaffected by genistein
(Figure 6;
Table 5). This might be
explained by high concentrations (2 µM) of cGMP required to increase
tyrosine phosphorylation in human spermatozoa
(Willipinski-Stapelfeldt et al,
2004). NO (and by inference, cGMP) produced by SNAP is much higher
than that produced by PPCM (Table
2).
Pathway Leading to NO-Induced AL May Depend on Location and Concentration of NO
The pathway activated in response to NO is likely determined by the
concentration and intracellular location of NO. NO production in response to
PPCM is relatively low (Table
2) and likely occurs at or near the site of initial interaction of
PPCM with the plasma membrane. NO production is mediated by eNOS
(Figure 1), a particulate or
membrane form of the enzyme. In contrast, NO produced from SNAP occurs at much
higher levels (Table 2). NO
release from SNAP likely occurs in a more diffuse fashion, allowing for the
opportunity to interact with a wider array of intracellular effector elements.
However, relatively localized release of NO from NOSPPA-23 resulting from a
vector-mediated delivery could explain the greatly increased efficacy of this
compound as a stimulus of AL.
Mechanism-Based Design of Compound as a More Efficacious Stimulus of AL
Discovery of better second-generation compounds can be approached through
structural modifications of the parent compound, based on its intracellular
mechanism(s) of action. The proposed intracellular mechanism of PAL formed the
basis for the design and synthesis of NOSPPA-23.
We propose that the PPCM moiety of NOSPPA-23 acts as a vector for the targeted delivery of NO. As part of the delivery system, the sperm-selective properties of the vector are enhanced. NOSPPA-23 was predicted to: 1) induce AL in the presence or absence of Ca2+, because of the Ca2+-independent actions of the NO donor; 2) induce AL in the absence of Ca2+ at a lower concentration of NO donor equivalents than required for NO donor alone, because the source of NO is directed to the surface of the spermatozoon by the PPCM moiety; and 3) be synergistic compared with either NO donor or PPCM alone in the presence of Ca2+, because of different, but related, mechanisms by which the parent compounds induce AL (ie, an interactive effect is predicted).
Data presented in Figures 7 and 8 substantiate these predictions. NOSPPA-23, a prototype of an intramolecular combinatorial approach to an improved sperm-active compound, induces premature AL in either the presence or absence of added extracellular Ca2+. The response, either in the presence or absence of Ca2+, is synergistic to the predicted response to the NO donor and PPCM added in combination. The ED50 of NOSPPA-23 as a stimulus of AL in the presence of added extracellular Ca2+ (Figure 8, panel A) is substantially less than that of the NO donor from which it is derived, and 35-fold less than that of PPCM.
NO has known antimicrobial activity (Fang, 1997). Preliminary studies with NOSPPA-23 as an agent against HIV-1 and HSV-2 (Anderson, unpublished) suggest that covalent attachment of an NO donor to PPCM represents a viable approach to producing a contraceptive microbicide with improved biological activity.
The present study clearly shows synergistic activity of NOSPPA-23 regarding its ability to induce premature AL. NOSPPA-23 is only a single prototype of this new class of compounds, and may not represent the optimum vector (represented here by PPCM), NO donor (nitrooxypropanol, in this instance), or degree of substitution (where possible; 23% in the present study). Further work with this vector-assisted system for targeted microbicide enhancement is highly warranted, in which these variables are considered, and the range of in vitro and in vivo biological activities is examined.
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