Enhancement by elevated external potassium of the maximal responses to acetylcholine and norepinephrine in the rat vas deferens

In the rat vas deferens, the maximal response to acetylcholine increased almost linearly as a function of external potassium concentration, [K⁺]₀. The magnitudes of the maximal responses in 10, 15 and 20 mM [K⁺]₀ were 128, 155 and 184% of the maximal response in 6 mM [K⁺]₀, respectively. The response to carbachol also increased in 20 mM [K⁺]₀ to almost the same extent. The enhancement of the norepinephrine response was observed only in 20 mM [K⁺]₀; its magnitude was only 125%. The increase in [K⁺]₀ from 6 to 20 mM did not affect the sensitivity (expressed as the geometric mean ED₅₀) either to norepinephrine or to acetylcholine. Denervation of the postganglionic nerves to the vas deferens did not affect the enhancement of the maximal responses in 20 mM [K⁺]₀, indicating that the release of neuronal transmitters may not be involved.     

Bicarbonate-dependent action of Bay K 8644 in the smooth muscle of the rat vas deferens.

Effects of Bay K 8644, partial depolarization with high potassium, and nifedipine on the dose-response curves of the rat vas deferens to norepinephrine, methacholine and KCI were investigated in HEPES-buffered physiological salt solution (PSS) with or without 20 mM sodium bicarbonate. In the bicarbonate-containing PSS, Bay K 8644 at 10(-6) M enhanced the maximal contractions in response to norepinephrine, methacholine and KCI by 31.4, 103.3 and 40.1%, respectively. In the bicarbonate-free PSS, where the maximal contractions induced by norepinephrine, methacholine and KCI were 77.5, 75.0 and 68.2% of those in the bicarbonate-containing PSS, respectively. Bay K 8644 did not enhance the maximal contractile response to any of the agonists, although the contractions induced by low concentrations of KCI were increased by Bay K 8644. Increasing the potassium concentration in the PSS from 6 to 20 mM enhanced the maximal contractions in response to norepinephrine and methacholine in the bicarbonate-containing PSS, whereas in the bicarbonate-free PSS, the treatment decreased the contractions. In the two PSSs, nifedipine similarly inhibited the contraction in response to either 10(-5) M norepinephrine or 68 mM KCI. These results suggest that bicarbonate ion modulates the function of the voltage-dependent calcium channel in the smooth muscle of the rat vas deferens.     

Persistent enhancement of potassium-induced responses of the rat vas deferens by desipramine

Summary The effect of desipramine on the cumulative dose-response curves of noradrenaline and potassium (K⁺) was examined on the isolated rat vas deferens. An exposure of 10 min to 10^–7 M desipramine caused a leftward shift and an increase in the maximum response of cumulative dose-response curves of noradrenaline. Desipramine (10^–7 M), in contact with the tissue for 10 min, enhanced responses to cumulative additions of K⁺ without causing a consistent change in threshold concentrations. Wash-out of desipramine resulted in a rapid loss of enhanced maximum response to noradrenaline while the maximum response to K⁺ did not show any decrease for up to 120 min after wash-out of drug. One possible explanation for the persistent enhancement of K⁺-induced responses may be that desipramine causes postjunctional changes which selectively influence contractile responses of this tissue to K⁺.     

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force

Summary The relationship between Na⁺, K⁺-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb⁺ and Tl⁺ inhibited Na⁺, K⁺-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na⁺ concentrations. Tl⁺ produced a dose-dependent inhibition of Na⁺, K⁺-ATPase activity in the presence of 20 mM Na⁺ and 75 mM K⁺, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K⁺, Cs⁺, NH₄ ⁺, Na⁺ or Li⁺ produced essentially no effect on the Na⁺, K⁺-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K⁺ and 145 mM Na⁺), addition of Cs⁺ failed to alter the isometric contractile force significantly. NH₄ ⁺ and K⁺ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K⁺ was followed by a negative inotropic response. Rb⁺ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl⁺ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb⁺ and Tl⁺ were insensitive to propranolol pretreatment. Concentrations of Tl⁺ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na⁺-pump inhibition. The onset of the positive inotropic response to Rb⁺ or Tl⁺ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.     

Role of bicarbonate ion in the postjunctional action of cocaine in the smooth muscle of the rat vas deferens

In the HEPES-buffered physiological solution containing 20 mM bicarbonate ion, cocaine not only increased the sensitivity to norepinephrine but enhanced the maximal contractions to norepinephrine and methacholine in the rat vas deferens. Cocaine also potentiated the high potassium-induced contractions. In the solution without bicarbonate ion, such effects of cocaine were lost except for the sensitivity increase to norepinephrine. These results suggest that bicarbonate ion is crucial for the postjunctional stimulatory action of cocaine in the rat vas deferens.     

Contraction Induced Either by Iso-osmolar or Hyper-osmolar Potassium-rich Solutions Influences Relaxant Responses to Pinacidil and Verapamil in Rat Isolated Aorta

Abstract— Relaxant responses of pinacidil and verapamil were studied in rat isolated aorta contracted by either iso-osmolar or hyper-osmolar potassium-rich solutions. Relaxant response profiles of pinacidil and verapamil in rat isolated aorta contracted by 124 mm K⁺ hyper-osmolar Krebs solutions showed marked reductions in inhibiting E_max values and substantial increases in corresponding IC50 values in comparison with results obtained at iso-osmolar conditions. Changes in the slopes of the fitted log concentration-relaxation curves were also observed, whereas pinacidil relaxation curves obtained after initial contraction induced by 30 mm K⁺ Krebs solutions were only slightly influenced by osmolarity, a definite decrease in E_max occurred for verapamil at hyper-osmolar conditions. Initial tension development was much slower and maximal tension lower when induced by 124 mm K⁺ in hyper-osmolar compared with iso-osmolar Krebs solutions. After incubation in Ca²⁺-deprived EGTA-containing Krebs solutions the maximal tension produced by 124 mm K⁺ iso-osmolar Krebs solution in rat aorta was nearly 95% reduced, whereas it was only reduced by 50% at hyper-osmolar conditions. Hyper-osmolarity as established by direct addition of potassium chloride to Krebs solutions in order to induce contraction in vascular smooth muscle could influence the in-vitro action profile, potency and intrinsic activity of the two vascular relaxant drugs.     

Effect of ethanol on the kinetics of rat brain (Na+ + K+) ATPase and K+-dependent phosphatase with different alkali ions.

(Na⁺ + K⁺)-dependent ATPase [(Na + K)-ATPase] and K⁺-dependent p-nitrophenyl phosphatase [pNPPase] activities in rat brain heavy microsomal fractions were studied in the presence of 120 mM Na⁺ and varied concentrations of K⁺, Rb⁺, Cs⁺, Li⁺ or NH₄⁺. Scatchard and Hill plots indicated non-hyperbolicity (cooperativity) with all except Li⁺, which supported a considerably lower activity than any of the other ions tested. Addition of 0.22 M ethanol to the incubation mixtures produced a formally competitive inhibition of ATPase activity with K⁺, Rb⁺ and Cs⁺, a non-competitive inhibition with Li⁺, and a mixed inhibition with NH₄⁺. The changes in pNPPase activity generally followed a similar but less clear-cut pattern. The values of the Hill constants were not changed for either enzyme activity. The findings are interpreted as evidence that ethanol inhibits ATPase activity by inducing conformational changes which alter the consequences of ion binding to the various receptor sites.     

Presynaptic agonist effect of phentolamine in the rabbit vas deferens and rat cerebral cortex

Clonidine inhibited the electrically-induced twitch response of the rabbit and rat isolated vas deferens preparations and also the K⁺-evoked release of [³H]noradrenaline from rat cortical slices. This effect of clonidine was antagonized competitively by yohimbine. Phentolamine inhibited the electrically-induced twitch response of the rabbit, but not the rat, vas deferens and in low concentrations (<·1 μm ) also inhibited the K⁺-evoked release of [³H]NA from rat cortical slices. These inhibitory effects of phentolamine were antagonized by yohimbine in a competitive manner but were not antagonized by indoramin, an α₁-adrenoceptor antagonist. In the rabbit vas deferens, the effects of phentolamine were shown not to be due to the stimulation of β-, H_1-, H_2-, 5-HT- or muscarinic receptors. These results are consistent with the view that phentolamine can act as an agonist at presynaptic α₂-adrenoceptors in the rabbit vas deferens and rat cortex but not in the rat vas deferens.     

Organ culture of young rat vas deferens as an in vitro model for the study of denervation supersensitivity

To investigate whether organ culture is a suitable in vitro model for studying the mechanisms of denervation-induced supersensitivity, we cultured 1-week-old rat vas deferens for 3 days with a basic applied tension of 20 mg. Cultured muscles showed supersensitivity to norepinephrine and methacholine with concomitant elevation of the maximal response. To compare these changes with those caused by denervation, young rats were chemically denervated by injecting 6-hydroxydopamine, and consequent sensitivity changes were investigated. Denervated muscles showed non-specific supersensitivity to norepinephrine and methacholine but the maximal response did not increase. When these denervated muscles were organ-cultured, they showed no or only a slight increase in sensitivity to norepinephrine and methacholine, but the maximal response increased greatly. These observations led to the suggestion that the increase in sensitivity may be mediated through the same mechanisms as those for denervation supersensitivity. The elevation of the maximal response was suggested to be produced by the improvement of cell-to-cell conduction as well as some other unknown factor(s) probably specific to organ culture. Thus, it was concluded that organ-cultured 1-week-old rat vas deferens is a useful model to study the mechanisms of denervation supersensitivity.     

Possible role of disturbed Na+ homeostasis in mouse vas deferens contractile hyperreactivity after imunological sensitization

In the present study we have investigated the involvement of sensitized mice immunoglobulins and some electrophysiological alterations that participate to the antigenic sensitization-induced hyperreactivity of isolated mouse vas deferens. Active sensitization was performed by subcutaneous injection of egg albumen. Contractile responses to noradrenaline were isometrically recorded in the isolated vas deferens. Low external Na⁺-induced contractions and rapid cooling contractures were evaluated. Resting membrane potential (Er) and intracellular Na activity were measured in control and actively sensitized vas deferens by using conventional KCl-filled and Na⁺-sensitive microelectrodes respectively. Active sensitization-induced hyperreactivity to noradrenaline was reproduced by in vitro passive sensitization of control vas deferens with sensitized mice immunoglobulins. The inhibition of the nitric oxide synthesis by N-nitro-L-arginine methyl ester (L-NAME) did not change control vas deferens reactivity in vitro to noradrenaline and acetylcholine. Rapid cooling contractures, performed after lowering external Na⁺ concentration, were not altered by active sensitization. However, sensitization increased significantly the strength of the low external Na⁺-induced contractions. In control vas deferens Er was a mean of –49.2±0.3 mV (mean ±SEM). Sensitization resulted in reduction of Er by 14 mV. In sensitized preparations, relative insensitivity of Er to ouabain, external K⁺ removal and cooling were observed. The intracellular Na⁺ activity was increased by about 40% in sensitized vas deferens. It is concluded that sensitization-induced hyperreactivity is mediated by immunoglobulins and produced smooth muscle cells depolarisation. The low Er of sensitized muscle may be partly the result of an increase in membrane permeability to Na⁺ which could interfere with intracellular Ca²⁺ homeostasis. Received: 26 May 1998 / Accepted: 22 July 1998     

Influence of Osmolality of Solutions Used for K+ Contraction on Relaxant Responses to Pinacidil,Verapamil, Theophylline and Terbutaline in Isolated Airway Smooth Muscle

Abstract: Concentration-relaxation profiles for pinacidil, verapamil, terbutaline and theophylline were studied in guinea-pig trachealis contracted by two commonly applied techniques for K⁺ depolarization. All drugs were much less effective on contractions induced by hyperosmolar 124 mM K⁺ solution (added KCl) than on contractions elicited by an isoosmolar 124 mM K⁺ Krebs solution (substituted KCl). The maximal relaxant responses were (isoosmolar K⁺/hyperosmolar K⁺): pinacidil 100%/40%, verapamil 100%/60%, theophylline 100%/0%, terbutaline 50%/0%. Addition of mannitol to establish the same hyperosmolarity as with 124 mM KCl also produced contraction of guinea-pig trachealis. Concentration-relaxation curves for the drugs on mannitol-induced contractions had close resemblance to those obtained in hyperosmolar 124 mM K⁺ solution. When contraction was elicited by 30 mM K⁺, pinacidil showed seven times higher relaxant potency in hyperosmolar compared to isoosmolar solution, whereas the relaxant responses to verapamil, theophylline and terbutaline were not influenced by osmolality. When K⁺ depolarization is used as a tool for evaluation of drug action in airway smooth muscle, the two different techniques produce dissimilar results. The influence of hyperosmolarity pec se appears to be an important and unwanted feature when K⁺ depolarization is produced by addition of KCl.     

A comparison of the responses of the isolated vas deferens of the rat to field stimulation and to noradrenaline added cumulatively and non-cumulatively

The maxima, slopes and positions of cumulative and non-cumulative dose-response curves for noradrenaline were determined on the isolated vas deferens of the rat. Cumulative log dose-response curves were flatter and reached a lower maximum than the non-cumulative curves. However transformation of the responses to fractions of the maximal responses produced curves with identical slopes and only slight differences in positions. Field stimulation led to larger and more rapidly attained maximal responses than did the addition of exogenous noradrenaline. Responses to field stimulation applied at the plateau of the cumulative dose-response curves were similar to those occurring in the control situation. It is suggested that the effectiveness of noradrenaline in producing contraction of the vas deferens may be related to the rate at which its concentration rises at the effector cells.     

The effect of pretreatment with 6-hydroxydopamine on the norepinephrine concentration and sensitivity of the rat vas deferens.

Injection of 6-hydroxydopamine via the dorsal vein of the penis results in a marked depletion of the endogenous norepinephrine of the vas deferens. Seven days after pretreatment with 6-hydroxydopamine there is a shift to the left and increase in maxima of the dose-response curves for norepinephrine and methoxamine. The results indicate that pretreatment with 6-hydroxydopamine produces denervation of the vas deferens and that the in vitro tissue exhibits both prejunctional and postjunctional supersensitivity.     

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors,nimodipine and nitrendipine

Nimodipine (0.015 to 1.5 μM) increased Na⁺, K⁺-ATPase activity by 70–120% in isolated smooth muscle membranes. At 0.015 μM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na⁺, K⁺ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na⁺,K⁺-ATPase by nimopidine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca²⁺ entry but also to the stimulation of the Na^? pump.     

Effect of Roflumilast in airways disorders via dual inhibition of phosphodiesterase and Ca2+-channel

The bronchodilator effects of Roflumilast “a selective phosphodiesterase type-4 (PDE4)” inhibitor studied in this experimental protocol. The spiral strips of isolated guinea-pig tracheal chains mounted in organ bath and maintained in Krebs solution ventilated with carbogen at 32 °C and in Ca⁺⁺ restricted krebs solution. PDE inhibitory activity was evaluated by recording dose response curves using inhibitory effect of isoprenaline on CCh induced contractions. For confirmation of PDE inhibition the intracellular cAMP levels were also estimated. Roflumilast resulted a sharp inhibition in contractile responses of carbachol (CCh, 1 µM) and K⁺ (80 mM) and the results were almost similar to verapamil. In Ca⁺⁺ restricted Krebs solution, a rightward shift in the Ca⁺⁺ response curves observed in the tracheal chain strips which were pretreated with Roflumilast (0.001–0.003 mg/mL) and the maximum response was suppressed, similarly as with verapamil. PDE inhibitory effect of Roflumilast evaluated by recording dose-dependent (0.03–0.1 mg/mL) responses, the isoprenaline-induced inhibitory dose response curves shifted leftward similar to papaverine (PDE inhibitor). Pretreatment with Roflumilast exhibited elevated intracellular cAMP levels in tracheal strips. Findings of the experiment conclude bronchodilatory influence of Roflumilast via PDE and Ca⁺⁺ channel inhibition. Results of current experiment offers comprehensive mechanistic background of Roflumilast in future as therapeutic bronchodilator for hyperactive bronchial airway diseases.     

Regional difference in sympathetic neurotransmitter-and Ca2+ channel-mediated responses in rat vas deferens

• 1.1. Pharmacological properties were compared in the epididymal and prostatic portions of the rat vas deferens.
• 2.2. Contractile response to norepinephrine (NE) was larger in the epididymal portion, in spite of the smaller diameter in this region. In contrast, contraction evoked by ATP or α,β-methylene ATP (α,β-mATP) was larger in the prostatic portion. The sensitivities to NE but not to α,β-mATP are different in these portions.
• 3.3. ATP or NE facilitated the contraction induced by the other agonist, suggesting that they cooperatively elicit smooth muscle contraction. This cooperation was observed in both portions.
• 4.4. Neither the contraction elicited by the addition of Ca²⁺ to smooth muscle depolarized with 63.7 mM extracellular K⁺ nor the relaxation by nifedipine of the depolarized smooth muscle precontracted with 1.8 mM extracellular Ca²⁺ was regionally different. However, Bay k 8644 elicited contraction only in the epididymal portion. A combination of 5 mM K⁺ with Bay k 8644 also caused oscillatory contraction in the prostatic portion.
• 5.5. A radioligand binding study was performed using the microsomal fraction prepared separately from these portions. Both the dissociation constant and the maximum binding for ³H-nimodipine were smaller in the epididymal fraction than in the prostatic fraction.
• 6.6. These results suggest that 1. the NE-elicited contraction is more pronounced in the epididymal portion, 2. the purinoceptor-mediated contractions along the vas deferens are less heterogeneous, and 3. although the sensitivity to Bay k 8644 is higher in the epididymal portion, Ca²⁺ channel-mediated responses are not regionally different when they are fully activated.

     

α1- and β-Adrenoceptor-Mediated Increase in 86Rb+-Uptake in Isolated Cardiomyocytes from Adult Rat Heart: Evidence for Interaction between the Two Receptor Systems

Abstract: The aim of the present investigation was to study the effect of α₁- and β-adrenoceptor stimulation, alone and in combination, on potassium uptake in isolated ventricular cardiomyocytes from adult rat heart, using the potassium analogue ⁸⁶Rb⁺. The reliability of ⁸⁶Rb⁺ as a potassium analogue was also investigated. α₁-, β-, And combined adrenoceptor stimulation was achieved by using noradrenaline in the presence and absence of appropriate adrenoceptor antagonists. The uptake of ⁸⁶Rb⁺ was found to increase linearly with time up to 20 min., both during basal and receptor-stimulated conditions. The basal uptake rate was about 0.18 ml/g protein X min. At 15 min. both α₁, β- and combined adrenoceptor stimulation dose-dependently increased the ⁸⁶Rb⁺-uptake with a -logEC₅₀ of 7.05, 6.68 and 6.73, respectively. The maximal increase in these series achieved by 5×10^?5 mol/l noradrenaline was 29%, 24% and 41% above basal level, respectively. Comparison of the maximal effects in the same cell preparations, with the observed value for combined adrenoceptor stimulation in each experiment as 100%, gave a relative maximal increase in ⁸⁶Rb⁺-uptake after separate α₁-adrenoceptor stimulation of 67±8%, and of 68±6% after separate β-adrenoceptor stimulation. The theoretically calculated value for combined adrenoceptor stimulation, if additivity, was 135±11%, which was significantly higher than the observed value (100%) (P=0.026). The effect of noradrenaline was not limited by the maximal ⁸⁶Rb⁺-uptake capacity, as 10^?5 mol/l forskolin increased the ⁸⁶Rb⁺-uptake more than noradrenaline. Examining the reliability of ⁸⁶Rb⁺ as potassium-analogue by combining ⁴²K⁺ and ⁸⁶Rb⁺ in the same experiments, showed that combined adrenoceptor stimulation dose-dependently increased both the ⁴²K⁺- and ⁸⁶Rb⁺-uptake with the same potency and to the same extent. Thus ⁸⁶Rb⁺ is a reliable potassium-analogue for these effects. In conclusion both α₁ and β-adrenoceptor stimulation dose-dependently increased the cellular ⁸⁶Rb⁺-uptake to the same extent and with the same potency. The observed maximal ⁸⁶Rb⁺-uptake after combined adrenoceptor stimulation was significantly higher than the maximal effect after either form of separate receptor stimulation, but significantly lower than expected if the effects were purely additive. The results thus show inhibitory interaction between the two receptor systems.     

Concentration-Response Relationship of α1-Adrenoceptor-Stimulated Increase of 86Rb+ Efflux in Rat Heart*

Abstract: The aim of the present study was to establish a concentration-response relationship for the α₁-adrenoceptor mediated increase of ⁸⁶Rb⁺ efflux, and to characterize the sensitivity of this response to the selective α₁-adrenoceptor antagonist prazosin. Isolated rat hearts were perfused retrogradely at constant flow and at 31°. Timolol (10^?6 mol/l) was used to block β-adrenoceptors. After a loading period with ⁸⁶Rb⁺ and 55 min. washout, the hearts were exposed to phenylephrine in a concentration range from 3×10^?8 mol/l to 10^?4 mol/l. Control experiments comparing the effects of α₁-adrenoceptor stimulation on ⁸⁶Rb⁺ efflux and ⁴²K⁺ efflux were performed. α₁-Adrenoceptor stimulation increased the ⁸⁶Rb⁺ efflux with a pD₂=6.35±0.20 (mean±S.E.M.). The maximal response to phenylephrine was 22.5±2.0% (mean±S.E.M.) of the control values. The concentration-response curve was shifted to higher concentration of agonist in the presence of the α₁-adrenoceptor antagonist prazosin (3× 10^?10 mol/l). The calculated inhibition constant for prazosin was 6.1×10^?11 mol/l. ⁸⁶Rb⁺ was found to be a suitable K⁺ analogue in the study of relative changes in K⁺ efflux although the basal efflux kinetics were different for the two isotopes. Conclusion: Phenylephrine increased the ⁸⁶Rb⁺ efflux concentration-dependently. A high sensitivity to prazosin confirmed the involvement of the α₁-adrenoceptor population.     

Binding of the alkali metal cations to tetracycline.

Cation binding by tetracycline is studied in aqueous solution at pH 2.3 and 8.6, using sodium-23 nuclear magnetic resonance. It is possible to determine the binding constants not only for the sodium but also, through competition experiments, for the other alkali metal cations. While no binding occurs at pH 2.3, at pH 8.6 complexation follows the sequence: Li⁺ > Na⁺, Cs⁺ > K⁺ > Rb⁺. The dissociation constants for the 1:1 complexes between tetracycline and the univalent alkali metal cations are of the order of 50 mM. Hence, the antibiotic is complexed by metal cations (Mg²⁺, K⁺ and, to a lesser extent, Na⁺) when it binds to the ribosome.