Introduction

Top Abstract Introduction Methods Results Discussion References

Vasopressin (AVP) causes powerful constriction in a variety of vascular regions through V₁ receptor activation and promotes reabsortion of water in renal tubular cells through V₂ receptors coupled to adenylate cyclase activation (Michell et al., 1979; Penit et al., 1983). With regard to human vessels, vasopressin causes powerful V₁ receptor mediated constriction in isolated mesenteric, (Martínez et al., 1994b; Ohlstein and Berkowitz, 1986) cerebral (Lluch et al., 1984; White and Robertson, 1987) and renal (Medina et al., 1996) arteries. Vasopressin may also modify the effects of other vasoactive substances that are found in plasma or released from perivascular nerve endings (Bartelstone and Nasmyth, 1965; Karmazyn et al., 1978; Guc et al., 1992). In the human forearm AVP attenuates phenylephrine-induced vasoconstriction (Harada et al., 1991) whereas recent experiments in human isolated mesenteric arteries show that AVP enhances adrenergic mediated responses (Medina et al., 1997).

The presence of high concentrations of vasopressin in human testis (Nicholson et al., 1984), and in penile erectile tissue (Andersson et al., 1987) together with the pharmacological characterization of specific V₁ receptors in this tissue (Andersson et al., 1988; Maggi et al., 1989) suggest that the hormone is taken up and/or synthetized locally (Andersson et al., 1987). Vasopressin was found to contract isolated human corpus cavernosum and spongiosum and preparations of the cavernosal and deferential artery (Hedlund and Andersson, 1985; Andersson et al., 1987; Medina et al., 1996). At present there is no information concerning the effects and pharmacological receptors of vasopressin in human penile dorsal arteries and veins. Furthermore, the possibility exists that AVP could importantly affect neurogenic vascular tone if this peptide would facilitate sympathetic neurotransmission or sensitize the smooth muscle to the effects of norepinephrine. This might have important implications in understanding the increase in penile smooth muscle tone resulting from excessive sympathetic outflow or increased blood catecholamine levels (von Euler, 1964; Diederichs et al., 1991). An increase in smooth muscle tone would oppose the relaxation necessary for erection. Therefore we designed this study to examine the direct effects of vasopressin on isolated human penile dorsal artery and deep dorsal vein. The second aim of the present study was to establish whether low concentrations of vasopressin could modify the constrictor responses of these vessels to adrenergic stimulation, analyzing the receptor subtypes involved.

	Methods

Top Abstract Introduction Methods Results Discussion References

Penile dorsal arteries and deep dorsal veins were obtained from 20 multiorgan donors during procurement of organs for transplantation (age range: 17-71 years). The study was approved by the ethical committee of our institution. The vessels were immediately placed in chilled Krebs-Henseleit solution, and rings 3 mm long were cut under a dissecting microscope (Heerbrugg, Switzerland) for isometric recording of tension.

Two stainless steel L-shaped pins 100 µm in diameter were introduced through the lumen of the ring. One pin was fixed to the wall of the organ bath, while the other was connected to a force-displacement transducer (Grass FT03). Changes in isometric force were recorded on a Grass polygraph (model 7). Each ring was set up in a 4 ml bath containing modified Krebs-Henseleit solution of the following millimolar composition: NaCl, 115; KCl, 4.6; MgCl₂.6H₂O, 1.2; CaCl₂, 2.5; NaHCO₃, 25; glucose, 11.1 and disodium EDTA, 0.01. The solution was equilibrated with 95% O₂ and 5% CO₂ to give a pH of 7.3-7.4. Temperature was held at 37°C. To establish the resting tension for maximal force development, a series of preliminary experiments were performed on rings of similar length and outer diameter which were exposed repeatedly to 100 mM KCl. Basal tension was increased gradually until contractions were maximal. The optimal resting tension was 3.5 g for the artery and 3 g for the vein. The rings were allowed to attain a steady level of tension during a 2-3 hr accommodation period before testing. Functional integrity of the endothelium was confirmed routinely by the presence of relaxation induced by acetylcholine (10⁷-10⁶ M) or substance P (10⁹-10⁸ M) during contraction obtained with norepinephrine (10⁷-3 × 10⁷ M).

Following the equilibration period, concentration-response curves for vasopressin (10¹¹-10⁷ M) were obtained in paired rings in the absence and in the presence of the V₁ antagonist d(CH₂)₅Tyr(Me)AVP (10⁸-10⁶ M).

Electrical field stimulation was provided by a Grass S88 stimulator (Grass Instruments, Quincy, MA) via two platinum electrodes positioned on each side and parallel to the axis of the vessel ring. To assess the nature of the contractile responses and avoid direct stimulation of smooth muscle, frequency-response relationships were determined on a group of vessels in the presence and absence of 10⁶ M tetrodotoxin, following procedures previously described (Martínez et al., 1995, 1994a; Aldasoro et al., 1993). In summary, the protocol was designed to find the optimal stimulation parameters causing a contractile response that was completely eliminated by 10⁶ M tetrodotoxin. Stimulation was conducted at 15 V for 15 sec at frequencies of 0.5, 1 and 2 Hz. A pulse width of 0.2 msec was used. A period of 10-15 min was allowed between stimulations.

To study the effects of vasopressin and the V₂ agonist desmopressin on electrical field stimulation-induced responses, frequency-response relationships were determined in a separate group of experiments. After an initial set of stimulations, the vessel rings were consecutively incubated with increasing concentrations of vasopressin (10¹²-3 × 10¹¹ M) or desmopressin (10¹⁰-10⁸ M) for 10 min before another set of stimulations was given. As a control, four consecutive sets of stimulations were given to a group of untreated rings at identical intervals. Less than 10% variability in magnitudes of electrical field stimulation-induced contractions was observed for a given ring during four consecutive sets of control stimulations.

In another series of experiments, the rings were incubated with the V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (3 × 10⁹-10⁷ M) or the V₂ receptor antagonist [d(CH₂)₅,D-Ile²,Ile⁴,Arg⁸]-vasopressin (10⁸-10⁷ M) for 10 min and then exposed to vasopressin (10¹¹ M). Electrical field stimulation was obtained in these rings and the data compared with rings in the absence of antagonists.

To determine whether vasopressin could block the reuptake of norepinephrine and therefore enhance the neurogenic induced contractions, the reuptake blocker cocaine (10⁶ M) was used in some experiments 10 min before the addition of vasopressin.

Concentration-response curves for norepinephrine were determined in a cumulative manner. Control (in the absence of vasopressin) and experimental (in the presence of vasopressin) data were obtained from separate vascular preparations. Another group of rings were incubated with the V₁ antagonist (3 × 10⁸ M) before exposure to vasopressin and norepinephrine.

Drugs. The following drugs were used: tetrodotoxin, guanethidine, prazosin hydrochloride, norepinephrine hydrochloride, acetylcholine chloride, substance P, arginine vasopressin acetate salt, [(1-(-mercapto-,-cyclopentamethylenepropionic acid)-2-(O-methyl)-tyrosine, 8-arginine) vasopressin] (d(CH₂)₅Tyr(Me)AVP), deamino-8-D-arginine vasopressin (desmopressin), (Sigma Chemical Co, St. Louis, MO, U.S.A.), [d(CH₂)₅, D-Ile²,Ile⁴,Arg⁸]-vasopressin (Peninsula Laboratories Europe, Merseyside, England) and cocaine chlorhydrate (Abelló, Madrid, Spain). All drugs were dissolved in Krebs solution. Drugs were added to the organ bath in volumes of less than 70 µl. Stock solutions of the drugs were freshly prepared every day, and kept on ice throughout the experiment.

Data analysis. All values are expressed as mean ± S.E. Contractions are reported as a percentage of response to KCl (100 mM). pD₂ (negative logarithm of the molar concentration at which half-maximum contraction occurs) was determined from individual concentration-response curves by nonlinear regression analysis. The pA₂ values for V₁ vasopressin receptor antagonist were determined from a Schild plot (Arunlakshana and Schild, 1959). The concentration ratios (CR) were calculated as the ratio between the EC₅₀ value (concentrations producing half-maximal contractions) for vasopressin in the presence and absence of different concentrations of the antagonist. A Schild plot was constructed with the CRs: log (CR-1) (ordinate scale) was plotted against log (antagonist concentration) (abscissa scale) and pA₂ was estimated as the intercept of the regression line with the abscissa scale (Arunlakshana and Schild, 1959). Concentration-response curves of the tested agonists or frequency-response relationships were performed in rings obtained from the same patient; the responses obtained in each patient were averaged to yield a single value. Therefore, all n values are presented as the number of individuals from whom the rings were obtained. For electrical stimulation experiments, in which the same rings were stimulated in the absence and presence of antagonists, a paired t test was used. Statistically significance was accepted at P < .05.

	Results

Top Abstract Introduction Methods Results Discussion References

Effects of vasopressin. Vasopressin (10¹¹-10⁷ M) caused concentration-dependent contractions in arteries and veins (fig. 1). The maximal contractions to vasopressin and pD₂ values are shown in table 1. The presence of the V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP (10⁸-10⁶ M) induced significant shifts (P < .05) of the control curve to the right in a concentration-dependent manner, but differences in the maximal tensions developed were not significant (fig. 1). The ₁ adrenoceptor blocker prazosin (10⁶ M) did not affect the concentration response curves to vasopressin (fig. 1). The pA₂ and slope values obtained for the V₁ antagonist in arteries and veins are shown in table 1.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Concentration-response curves for vasopressin in dorsal arteries and deep dorsal veins in the absence (), and in the presence of V1 antagonist d(CH2)5Tyr(Me)AVP (, 108 M; , 107 M; , 106 M) or in the presence of the 1 adrenergic antagonist prazosin (, 106 M).

11

7

7

Effects of vasopressin on electrical stimulation -induced responses. Electrical stimulation induced frequency-dependent increases in tension in both arteries and veins which were abolished by tetrodotoxin, guanethidine and prazosin (all at 10⁶ M), thus indicating that the effect was due to the release of norepinephrine from adrenergic nerve endings acting on alpha-1 adrenoceptors.

12

11

11

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Top. Original tracings of contractile responses to electrical field stimulation (1 Hz) of human penile dorsal artery and deep dorsal vein under control conditions and after incubation with various concentrations of vasopressin (1012 to 3 × 1011 M). Bottom. Bar graphs of contractile responses to electrical stimulation (1 Hz) in the absence and in the presence of vasopressin. * P < .05 vs. control.

9

7

8

11

7

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inhibition by the V1 antagonist d(CH2)5Tyr(Me)AVP (3 × 109-107 M) of the potentiation induced by 1011 M vasopressin on electrical stimulation-induced responses.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Bar graphs showing effects of 1011 M vasopressin on frequency-dependent contractile responses to electrical field stimulation in the absence and in the presence of 3 × 108 M V1 antagonist d(CH2)5Tyr(Me)AVP. *P < .05 vs. control.

10

8

11

8

7

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Contractile responses to electrical stimulation in the absence and in the presence of increasing concentrations (1010-108 M) of the V2 receptor agonist desmopressin.

6

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Frequency-response relationship in the absence and in the presence of cocaine (106 M) or cocaine together with vasopressin (1011 M) *P < .05 vs. control.

Effects of vasopressin on norepinephrine induced contraction. Norepinephrine induced concentration dependent contraction in penile arteries and veins (fig. 7). In the presence of vasopressin (3 × 10¹¹ M) the concentration response curves to norepinephrine were displaced to the left without changing maximal contractions. The V₁ receptor antagonist (3 × 10⁸ M) completely reversed the vasopressin-induced potentiation (fig. 7 and table 2).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Concentration-response curves to norepinephrine in the absence () and in the presence of vasopressin (, 3 × 1011 M) and in the presence of the V1 receptor antagonist (3 × 108 M) together with vasopressin ().

	Discussion

Top Abstract Introduction Methods Results Discussion References

Our experiments provide the first evidence for the powerful constrictor action of vasopressin in penile deep dorsal artery and vein. Antagonists of arginine vasopressin have been used to study the vascular effects of this peptide and to characterize the receptors involved (Sawyer et al., 1981). Furthermore, these antagonists have been reported to be potent inhibitors of the contractile response of human corpus spongiosum to vasopressin (Andersson et al., 1987). We demonstrate that d(CH₂)₅Tyr(Me)AVP inhibited the vasopressin contraction in a competitive way over a given concentration range of the antagonist. Schild analysis showing unitary slopes and antagonist pA₂ values obtained from these data indicate that the receptors involved in vasopressin induced contraction belong to the classical V₁ receptors (Sawyer and Manning, 1985). Similar pA₂ values for the same V₁ antagonist have been found in human uterine arteries (Jovanovic et al., 1995) and in several vascular beds of the rabbit (García-Villalón et al., 1996).

The present study also shows that low concentrations of vasopressin enhance the contractile effects of electrical stimulation and norepinephrine. The potentiating effects occur at vasopressin concentrations lower than those required to produce a clear direct contractile response.

We examined the potential role of V₂ receptor stimulation in the enhancing effects of vasopressin. The results do not support the intervention of V₂ receptors in these responses. First, the selective V₂ agonist desmopressin did not modify responses to vasopressin or to electrical field stimulation. On the other hand, the V₂ receptor antagonist [d(CH₂)₅,D,Ile²,Ile⁴,Arg⁸]vasopressin did not affect the potentiation induced by vasopressin. In contrast, the selective V₁ receptor antagonist d(CH₂)₅Tyr(Me)AVP inhibited the potentiating effects of vasopressin on electrical field stimulation and norepinephrine- induced contraction in a concentration-dependent manner. Therefore, the results exclude a role for V₂ receptors in the potentiating effects of vasopressin and they are consistent with the hypothesis that V₁ receptor stimulation by vasopressin in the absence of direct contraction is followed by enhancement of responses to both endogenous and exogenous norepinephrine.

It might be conceived that the effects of vasopressin on electrical stimulation contractions could involve an effect on adrenergic nerves leading to release of norepinephrine or alternatively, vasopressin could act with norepinephrine at postjunctional receptor sites. Because norepinephrine release was not measured in this study, a contribution of prejunctional facilitating effects cannot be excluded. The fact that the concentration response curves to vasopressin were not modified by prazosin, an ₁-adrenoceptor blocker, suggests that the action of this peptide does not involve release of norepinephrine. The possibility that vasopressin could block the reuptake of norepinephrine and therefore enhance the contractile response is unlikely because the potentiating effects were still evident in the presence of cocaine. In the conditions of our experiments, cocaine per se failed to potentiate the vasoconstriction produced by nerve stimulation, a finding similar to that recently observed in human saphenous vein (Medina et al., 1998). This suggests that neuronal reuptake of norepinephrine in these vessels is of little importance, a circumstance that is mainly dependent on vascular region and species (Berkowitz et al., 1971; De la Lande et al., 1967; Lluch et al., 1975).

It has been shown that human corpus cavernosum and corpus spongiosum contain vasopressin in concentrations higher than those normally found in the circulation (Andersson et al., 1987). This was interpreted to indicate that the hormone might be synthetized locally or taken up and stored (Kasson and Hsueh, 1986; Andersson et al., 1987). Such a circumstance, together with the potent contractile effects of vasopressin on penile vessels may lead to speculate that vasopressin may act as a cotransmitter. If vasopressin is released together with norepinephrine from adrenergic nerves and contributes to the contractile effects of nerve stimulation, V₁ antagonists should partially block these contractile effects. The V₁ antagonist effectively blocked vasopressin contractions, but electrically-induced contractions were unaffected. Another possible explanation for the vasopressin induced potentiation is a change at the receptor level leading to an increased affinity of norepinephrine for its receptor. This may be a likely explanation, because vasopressin increased the contractions to exogenous applied norepinephrine. Thus our data are consistent with the suggestion that potentiation of the effects of nerve stimulation by vasopressin corresponds to a postsynaptic enhancement of the action of norepinephrine.

Stimulation of the lumbar sympathetic chain produces detumescence or inhibition of erection in various animal species (Carati et al., 1987; Diederichs et al., 1991; Giuliano et al., 1993). Therefore the flaccid state of the penis has been considered to depend on activation of adrenergic nerves. Although vasopressin does not seem to act as a co-transmitter with norepinephrine in these vessels, the recent discovery that vasopressin may be synthetized by vascular smooth muscle cells (Simon and Kasson, 1995) raises the possibility that locally released vasopressin may reach concentrations high enough to induce penile vasoconstriction and act synergistically with the adrenergic neurotransmitter. The concentrations of vasopressin in this study would be expected to be similar to basal plasma concentrations in normal humans (Harada et al., 1991; Hirsch et al., 1989) and lower than those observed in response to hypotension, dehydration, exercise, and in some patients with hypertension or congestive heart failure (Melin et al., 1980; Nicod et al., 1985; Sorenson and Hammer, 1985). Consequently, the direct contractile effects of vasopressin together with its amplifying effects on adrenergic-mediated constriction should be taken into consideration in the overall regulation of penile erection and in those states characterized by increased plasma vasopressin levels.