Presence Of Bay K8644 Research Articles

Comparison of Ca2+ Handling for the Regulation of Vasoconstriction between Rat Coronary and Renal Arteries

Background: Ca²⁺ plays an important role in the regulation of vasoconstriction. Ca²⁺ signaling is regulated by a number of Ca²⁺-handling proteins. However, whether differences in Ca²⁺ handling affect the regulation of vasoconstriction in different arteries remains elusive. Objective: To determine whether differences in Ca²⁺ handling affect the response to vasoconstrictors in different arteries. Methods: Arterial ring contraction was measured using a Multi Myograph System. Vascular smooth muscle cells (VSMCs) were digested with type 2 collagenase in DMEM, then intracellular calcium concentration was measured with the Ca²⁺ probe fluo-4/AM in the isolated cells. Calcium-related proteins were assayed by Western blotting. Results: Phenylephrine did not induce ­coronary arterial contraction. There were differences in ­5-hydroxytryptamine, 9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F_2a, and endothelin 1-induced vasoconstriction in different solutions between coronary and renal arteries. Vasoconstrictions in the presence of Bay K8644 were stronger in coronary than in renal arteries. Store-operated calcium (SOC) channels could mediate Ca²⁺ influx in VSMCs of both groups. SOC channels did not participate in the contraction of coronary arteries. In addition, there were significant differences in the expressions of receptors and ion channels between the two groups. Conclusions: Ca²⁺ handling contributed to the different responses to vasoconstrictors between coronary and renal arteries.

Effect of the intracellular calcium concentration chelator BAPTA acetoxy-methylester on action potential duration in canine ventricular myocytes.

Intracellular calcium concentration ([Ca2+]i) is often buffered by using the cell-permeant acetoxy-methylester form of the Ca2+ chelator BAPTA (BAPTA-AM) under experimental conditions. This study was designed to investigate the time-dependent actions of extracellularly applied BAPTA-AM on action potential duration (APD) in cardiac cells. Action potentials were recorded from enzymatically isolated canine ventricular myocytes with conventional sharp microelectrodes. The effect of BAPTA-AM on the rapid delayed rectifier K+ current (IKr) was studied using conventional voltage clamp and action potential voltage clamp techniques. APD was lengthened by 5 μM BAPTA-AM - but not by BAPTA - and shortened by the Ca2+ ionophore A23187 in a time-dependent manner. The APD-lengthening effect of BAPTA-AM was strongly suppressed in the presence of nisoldipine, and enhanced in the presence of BAY K8644, suggesting that a shift in the [Ca2+]i-dependent inactivation of L-type Ca2+ current may be an important underlying mechanism. However, in the presence of the IKr-blocker dofetilide or E-4031 APD was shortened rather than lengthened by BAPTA-AM. Similarly, the APD-lengthening effect of 100 nM dofetilide was halved by the pretreatment with BAPTA-AM. In line with these results, IKr was significantly reduced by extracellularly applied BAPTA-AM under both conventional voltage clamp and action potential voltage clamp conditions. This inhibition of IKr was partially reversible and was not related to the Ca2+ chelator effect BAPTA-AM. The possible mechanisms involved in the APD-modifying effects of BAPTA-AM are discussed. It is concluded that BAPTA-AM has to be applied carefully to control [Ca2+]i in whole cell systems because of its direct inhibitory action on IKr.

The calcium channel agonist BAY K8644 enhances the responsiveness of human airway muscle to KCl and histamine but not to carbachol.

The role of calcium channel antagonists in asthma remains controversial. A new compound BAY K8644 is an agonist at the calcium voltage-dependent channel (VDC). Therefore, we studied the effect of BAY K8644 on contraction to KCl, histamine, and carbachol in human bronchus in vitro to assess the role of the VDC in this tissue. Cumulative concentration response curves were obtained to the agonists in bronchial spiral strips in the absence and in the presence of BAY K8644. Contractile responses to histamine were significantly (p less than 0.05) enhanced by BAY K8644 (10(-6) M) at concentrations between 10(-7) M and 3 X 10(-6) M and there was a significant decrease in the histamine EC50 (the concentration producing half the maximal response). Responses to low concentrations of KCl (10(-4) M to 3 X 10(-3) M) were also potentiated. BAY K8644 did not affect carbachol-induced contractions. We conclude that calcium influx through VDC occurs in response to KCl and histamine but not to carbachol. Such findings could explain the variability in efficacy of calcium channel antagonists in inhibiting various forms of bronchospasm.

Cytosolic calcium changes affect the incidence of early afterdepolarizations in canine ventricular myocytes.

This study was designed to investigate the influence of cytosolic Ca(2+) levels ([Ca(2+)]i) on action potential duration (APD) and on the incidence of early afterdepolarizations (EADs) in canine ventricular cardiomyocytes. Action potentials (AP) of isolated cells were recorded using conventional sharp microelectrodes, and the concomitant [Ca(2+)]i was monitored with the fluorescent dye Fura-2. EADs were evoked at a 0.2 Hz pacing rate by inhibiting the rapid delayed rectifier K(+) current with dofetilide, by activating the late sodium current with veratridine, or by activating the L-type calcium current with BAY K8644. These interventions progressively prolonged the AP and resulted in initiation of EADs. Reducing [Ca(2+)]i by application of the cell-permeant Ca(2+) chelator BAPTA-AM lengthened the AP at 1.0 Hz if it was applied alone, in the presence of veratridine, or in the presence of BAY K8644. However, BAPTA-AM shortened the AP if the cells were pretreated with dofetilide. The incidence of the evoked EADs was strongly reduced by BAPTA-AM in dofetilide, moderately reduced in veratridine, whereas EAD incidence was increased by BAPTA-AM in the presence of BAY K8644. Based on these experimental data, changes in [Ca(2+)]i have marked effects on APD as well as on the incidence of EADs; however, the underlying mechanisms may be different, depending on the mechanism of EAD generation. As a consequence, reduction of [Ca(2+)]i may eliminate EADs under some, but not all, experimental conditions.

L-type Ca2+ channel blockers inhibit the window contraction of mouse aorta segments with high affinity

L-type calcium channel blockers (LCCBs) reduce blood pressure more effectively in hypertensive than in normotensive subjects and are more effective in vascular smooth muscle (VSM) than in cardiac muscle. This has been explained by the depolarized resting potential of VSM in comparison with heart muscle cells and during hypertension, because both favor the “high affinity” inactivated state of the L-type calcium channel (LCC). Depolarized resting potentials, however, also increase Ca2+ influx via window, non-inactivating LCC. The present study investigated whether these channels can be effectively blocked by nifedipine, verapamil or diltiazem, as representatives of different LCCB classes. C57Bl6 mouse aortic segments were depolarized by 50mM K+ to attain similar degree of inactivation. The depolarization evoked biphasic contractions with the slow force component displaying higher sensitivity to LCCBs than the fast component. Removal of the fast force component increased, whereas stimulation of Ca2+ influx with the dihydropyridine BAY K8644, a structural analog of nifedipine, decreased the efficacy of the LCCBs. Addition of LCCBs during the contraction caused concentration-dependent relaxation, which was independent of the presence of a fast force component, but still showed lower sensitivity in the presence of BAY K8644. Our data suggest that steady-state contractions by depolarization with 50mM K+ are completely due to window Ca2+ influx, which is preferentially inhibited by LCCBs. Furthermore, results point to interactions between the LCCB receptors and Ca2+ ions or BAY K8644. The high affinity for open, non-inactivating LCC may play a dominant role in the anti-hypertensive effects of LCCBs.

Sarcoplasmic reticulum Ca2+ refilling is determined by L-type Ca2+ and store operated Ca2+ channels in guinea pig airway smooth muscle

Sarcoplasmic reticulum Ca2+ refilling (SRREF) is crucial to sustain the agonists induced airway smooth muscle contraction. Nevertheless, its mechanisms have not been clearly described yet, although L-type voltage dependent, store operated, receptor operated channels and the Na+/Ca2+ exchanger in its reverse mode (NCXREV) have been proposed as Ca2+ handling proteins participating in this process. We found that in guinea pig and bovine tracheal smooth muscle, SRREF induced by caffeine was completely abolished by thapsigargin, even in the presence of Bay K8644, an activator of the L-type Ca2+ channel. Activation of NCXREV in guinea pig tracheal myocytes increased SRREF in ~70%, while opening of the L-type Ca2+ channels with Bay K8644 and favoring the capacitative Ca2+ entry with 2-APB (32μM) also augmented the SRREF by ~170% and ~71%, respectively. Methoxyverapamil (D-600, an L-type Ca2+ channel blocker), 2-APB (100µM, antagonist of the capacitative Ca2+ entry) and PPADS (NCXREV blocker) diminished the SRREF by ~63%, ~72% and ~31%, respectively. The simultaneous addition of D-600 and 2-APB annulled SRREF. These last results were also seen when carbachol was used instead of caffeine. In tracheal rings, 2-APB and nifedipine abolished the carbachol-induced contraction. We concluded that the sarcoplasmic reticulum Ca2+ pump is the only mechanism involved in the SRREF and that L-type Ca2+ voltage dependent and store operated Ca2+ channels are the principal membranal Ca2+ handling proteins that provide extracellular Ca2+ for SRREF and carbachol-induced contraction in the guinea pig airway smooth muscle; NCXREV seems to play a minor role.

κ-opioid Receptor Activation Prevents Against Arrhythmias by Preserving Cx43 Protein Via Alleviation of Intracellular Calcium

κ-opioid receptor (κ-OR) activation with U50,488H, a selective κ-OR agonist, has been previously demonstrated to prevent against cardiac arrhythmias via stabilizing the synthesis and degradation of an integral membrane protein, Cx43, in gap junctions. However, the exact prevention mechanism remains unclear. The present study tested the hypothesis that the kappa OR agonist U50,488H mediates the prevention of arrhythmia through the regulation of intracellular calcium leading to the preservation of Cx43 protein. By performing electrocardiogram monitoring and immunoblotting in isolated Langendorff-perfused rat hearts, high concentrations of calcium-perfused rat hearts exhibited increased cardiac arrhythmias. Diminished expression of Cx43 protein was observed. The utilization of a whole-cell patch clamp technique revealed that U50,488H inhibited L-type calcium current in single ventricular myocytes in a dose-dependent manner. These effects were blocked by nor-binaltorphimine, potent and selective κ-OR antagonists. Administration of U50,488H before myocardial ischemia resulted in an attenuated of total arrhythmia scores. The attenuation effect was blocked by nor-binaltorphimine. The attenuation effect was antagonized both by Bay K8644, a L-type calcium channel agonist, and also by the Cx43 uncoupler heptanol. Finally, immunoblotting data demonstrated that the preservation of Cx43 protein conferred by U50,488H was reversed in the presence of Bay K8644. In summary, the present study demonstrates κ-OR activation with U50,488H may confer antiarrhythmic effects via modulation of the calcium-Cx43 pathway.

Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats

This study aimed to investigate the effect of magnolol (5,5′-diallyl-2,2′-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca2+ currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3–100μM). In the presence of Bay K8644 (100nM), magnolol (10–100μM) inhibited the contraction induced by 10μM ACh. By contrast, tetrodotoxin (100nM) and Nώ-nitro-l-arginine methyl ester (l-NAME 100μM) did not change the inhibitory effect of magnolol (10μM). In addition, magnolol (3–100μM) inhibited the L-type Ca2+ currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca2+ channel activity.

Inhibition of the Cardiac L-Type Calcium Channel Current by Antidepressant Drugs

Antidepressants inhibit many membrane receptors and ionic channels, including the L-type calcium channel. Here, we investigated the inhibition of calcium current (I(Ca)) by antidepressants in enzymatically isolated rat ventricular myocytes using whole-cell patch clamp. The molecular mechanism of inhibition was studied by comparing the voltage and state dependence of antidepressant inhibition of I(Ca) to the respective properties of calcium antagonists, and by studying the effect of (+/-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) or diltiazem on the inhibitory potency of the antidepressants. All selected antidepressants inhibited calcium currents reversibly and concentration-dependently. At a stimulation frequency of 0.33 Hz, the antidepressants imipramine, clomipramine, desipramine, amitriptyline, maprotiline, citalopram, and dibenzepin blocked I(Ca), with IC(50) values of 8.3, 11.6, 11.7, 23.2, 31.0, 64.5, and 364 muM. The antidepressant drugs shifted steady-state inactivation curves of I(Ca) to negative voltages. The extent of the shift was similar to that induced by diltiazem or verapamil, but it was significantly smaller than that induced by felodipine. The use-dependent component of the antidepressant-induced block was similar to that of diltiazem, and it was significantly more and less, respectively, than those of felodipine and verapamil. In the presence of Bay K8644, antidepressants were more effective in inhibiting I(Ca). However, the inhibitory effect of antidepressants was also augmented by diltiazem, suggesting that these drugs do not compete with diltiazem for a single binding site. These data suggest that antidepressants exert their inhibitory action on cardiac L-type calcium channels by a specific interaction at a receptor site similar to, but distinct from, the benzothiazepine site.

A role for chromogranin A (4–16), a vasostatin-derived peptide, on human colonic motility. An in vitro study

The hypothesis that CgA-derived peptides may be involved in mechanisms modulating motility was tested. Human colonic smooth muscles were studied using an organ bath technique. Acetic acid (AA) effects were characterized on spontaneous mechanical activities (SMA) and on responses to transmural nerve stimulation (NS). AA induced a significant decrease in tone and abolished SMA; this effect was insensitive to either TTX or l-NAME/apamin. The AA-induced inhibitory effects were significantly reduced in the presence of CgA4–16. This effect was insensitive to TTX or l-NAME/apamin. Furthermore, AA-induced effects were blocked in the presence of BAYK8644 and CgA4–16 together. The inhibitory effect of nifedipine was delayed in the presence of CgA4–16. NS induced a triphasic response. Only the excitatory components were reduced in the presence of AA. This effect was dose-related and remained unchanged in the presence of CgA4–16 alone, but was blocked in the presence of simultaneous administration of CgA4–16 and l-NAME/apamin. AA application induced inhibition of human colon motility in vitro. This effect may be mediated through an action on L-type calcium channels. CgA4–16 may display a protective role, which prevents the inhibition of motility due to AA to occur, by acting on both smooth muscle and afferent terminals.

Catecholamine release from cultured bovine adrenal medullary chromaffin cells in the presence of 60-Hz magnetic fields

Effects of powerline frequency (50/60 Hz) electric and magnetic fields on the central nervous system may involve altered neurotransmitter release. This possibility was addressed by determining whether 60-Hz linearly polarized sinusoidal magnetic fields (MFs) alter the release of catecholamines from cultured bovine adrenal chromaffin cells, a well-characterized model of neural-type cells. Dishes of cells were placed in the center of each of two four-coil Merritt exposure systems that were enclosed within mu-metal chambers in matched incubators for simultaneous sham and MF exposure. Following 15-min MF exposure of the cells to flux densities of 0.01, 0.1, 1.0 or 2 mT, norepinephrine and epinephrine release were quantified by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. No significant differences in the release of either norepinephrine or epinephrine were detected between sham-exposed cells and cells exposed to MFs in either the absence or presence of Bay K-8644 (2 μM) or dimethylphenylpiperazinium (DMPP, 10 μM). Consistent with these null findings is the lack of effect of MF exposure on calcium influx. We conclude that catecholamine release from chromaffin cells is not sensitive to 60-Hz MFs at magnetic flux densities in the 0.01–2 mT range.

Magnesium and cardiovascular disease.

Magnesium and cardiovascular disease.

Calcium dependent positive inotropic effects of low phorbol ester concentrations in isolated rat hearts

The aim was to examine the cardiac effects of phorbol esters over a wide concentration range and to determine if the effects are related to Ca2+ availability. Studies were carried out using isolated rat hearts exposed for 60 min either to phorbol 12-myristate 13-acetate (PMA, 10(-11) to 10(-6) M) or phorbol 12,13-dibutyrate (PDBu, 10(-12) to 10(-7) M) in the presence of either 1.25 or 2.50 mM CaCl2. Experiments were also done to assess the effect of BAY K8644, a Ca2+ agonist, on phorbol ester effects. After treatment, hearts were freeze clamped for later analysis of energy products. At the lowest concentrations studied, both PMA and PDBu produced positive inotropic effects, whereas higher concentrations resulted in a loss of contractile force in hearts perfused with 1.25 mM CaCl2. Doubling the CaCl2 concentration or the presence of BAY K8644 had little effect on the negative inotropic influence of either phorbol ester but reversed the positive inotropic effect to a negative inotropic one. Neither treatment had any effect on the coronary constricting effects of phorbol esters. Increases in resting tension and reductions in high energy phosphate content were evident only with the highest phorbol ester concentrations and were unaffected either by changes in CaCl2 concentrations or the presence of BAY K8644. At picomolar and nanomolar concentrations phorbol esters produce positive inotropic actions which are probably mediated by enhanced Ca2+ influx. Although higher concentrations produce negative inotropic effects, these are not influenced by [Ca2+]o nor are they related to disturbances in energy metabolism except at the highest concentrations. We conclude that phorbol esters produce complex concentration dependent cardiac effects which are not mediated by a single mechanism of action.

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye, fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca2+]i, was monitored with a fluorescent Ca2+ dye, fura-2. The fluorescence intensity ratio (I340/I380) was used as an index of [Ca2+]i. [Ca2+]i of the cells suspended in the nominally Ca(2+)-free solution was estimated at 52 +/- 3 nM. Ca2+ uptake was followed for as long as 5 min in the presence of 100-1000 microM added CaCl2. Most of the experiments were performed at 200 microM CaCl2. The Ca2+ uptake was abolished by 0.8 mM Ni2+ and 50 microM Mn2+ and partitally antagonized by 50 microM verapamil and 50 microM diltiazem but not affected by 20 microM nifedipine. The Ca2+ entry was reduced by increasing concentrations of extracellular K+ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca2+ transport doubled in the presence of Bay K8644 (8 microM), a Ca2+ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 microM nifedipine, 10 microM verapamil or 10 microM diltiazem and was relatively independent of extracellular K+ concentration. These results suggest that there are at least two distinct Ca2+ transport systems in the rat ileal epithelial cells, one resistant to organic Ca2+ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.

Effects of calcium and bay K-8644 on calcium currents in adrenal medullary chromaffin cells

The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca2+ currents, recorded in the presence of either 5.2 or 2.6 mM Ca2+ exhibited a single, noninactivating component. To analyze the effects of Ca2+ and Bay K-8644 on the kinetics of the Ca2+ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca2+] (2.5 mM) the midpoint of the steady-state Ca2(+)-channel activation curve lay at -6.9 mV. Increasing the [Ca2+] to 5.2 mM shifted the midpoint by -4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2 mM Ca2+) and 9.2 mV (for 2.5 mM Ca2+) induced an e-fold change in the activation of the current. Increasing [Ca2+]o from 2.5 to 5.2 mM induced a marked increase in the rate constant for turning on the Ca2+ permeability. Conductances were estimated from the slope of the linear part of the current-voltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5 mM Ca2+, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 microM increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 microM induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 microM) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from -6.9 to -13 mV. At the midpoint potential of -13 mV, a change in potential of 6.9 mV caused an e-fold change in Ca2+ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca2+ permeability.

Effect of BAY K8644 on cytosolic calcium transients and contraction in embryonic cardiac ventricular myocytes

Cytosolic calcium transients were recorded from spontaneously beating chick embryonic myocardial cell aggregates loaded with the fluorescent [Ca2+]i indicator, indo-1. Calcium transients rose rapidly from an end-diastolic [Ca2+]i of 230 +/- 18 nM to a peak systolic [Ca2+]i of 619 +/- 34 nM (n = 21). Relaxation of the transients was slow, and continued throughout diastole. Bay K8644 (0.5 microM) markedly prolonged the action potential and caused similar prolongation of the calcium transients. Calcium transients in the presence of Bay K8644 had an inflection on their rising phase, which was followed by a more gradual increase that continued until the membrane had repolarized to a negative potential of -15 to -30 mV. Bay K8644 caused marked elevation of peak systolic [Ca2+]i to 955 +/- 56 nM (P less than 0.002), with concomitant elevation of end-diastolic [Ca2+]i to 400 +/- 36 nM (P less than 0.002). Optical recordings of contraction showed changes similar to those in the calcium transient: the initial upstroke of the contraction was followed by a more gradual second component, which gave the contraction a "half-dome" appearance. The time to peak [Ca2+]i and the time to peak contraction increased linearly with action potential duration (APD50). The effects of Bay K8644 were simulated, in part, by CsCl (7.5 mM), which produced equivalent prolongation of the action potential and calcium transients. However, CsCl did not elevate diastolic [Ca2+]i. To determine the mechanism of the diastolic [Ca2+]i increase, Bay K8644 was applied to aggregates rendered quiescent by tetrodotoxin. Bay K8644 caused a graded increase in [Ca2+]i, which was followed by resumption of spontaneous beating.(ABSTRACT TRUNCATED AT 250 WORDS)

Proximal Upstream Flanking Sequences Direct Calcium Regulation of the Rat Prolactin Gene

Previous investigations have shown that Ca2+ strongly and specifically stimulates endogenous PRL gene expression by GH3 cells. In this study, addition of Ca2+ to Ca2+-deprived GH3 cells yielded a large (ca. 8-fold) stimulation of transient expression of a transfected PRL-chloramphenical acetyltransferase (CAT) construct containing ca. 1 kilo-base-pair of the PRL promoter region, but only a slight (less than or equal to 2-fold) nonspecific stimulation of CAT activity directed by any of three control promoters: dihydrofolate reductase, Rous sarcoma virus, or thymidine kinase. In GH3 cells never deprived of Ca2+, expression of a PRL-CAT construct was specifically stimulated and inhibited, respectively, by the dihydropyridine voltage-dependent Ca2+ channel modulators Bay K8644 and nimodipine; Ca2+ can thus regulate expression of an exogenous PRL promoter in cells incubated under physiological Ca2+ conditions. By employing a combined protocol, in which Ca2+-deprived cells are exposed to Ca2+ in the presence of Bay K8644, a very large (greater than 35-fold) but still promoter-specific induction of expression of a PRL-CAT construct was obtained. Analysis of 5'-deleted PRL-CAT constructs implied that the PRL gene Ca2+ response element is contained entirely within the first 174 base pairs of upstream flanking DNA sequence.

Tetrodotoxin‐resistant contractions induced by electrical stimulation of bladder muscle from man, rabbit and rat

Isolated detrusor preparations from man, rabbit and rat were suspended in an organ bath and isometric tension was recorded. The preparations were stimulated electrically in the presence of Bay K8644 and nifedipine before and after neuronal blockade with tetrodotoxin. Transmural electrical stimulation produced frequency-dependent contractions in all preparations. Bay K8644 significantly increased and nifedipine decreased these contractions. TTX effectively suppressed the response to electrical field stimulation in all species. When Bay K8644 was added to TTX blocked preparations, the responses to electrical stimulation were partly restored in bladder strips from man and rat. No increase in response was seen in the rabbit preparations. However, if the extracellular K+-concentration was increased to 10 mM (which per se did not affect the response) Bay K8644 significantly increased the contractions. All responses elicited by electrical stimulation in the presence of TTX were abolished by nifedipine. It is concluded that if the bladder smooth muscle is exposed to factors that can increase its sensitivity to contractile agents, this may result in uncontrolled (unstable) bladder contractions. Such contractions may use the 'normal' transmitter substances, but may be triggered at a lower stimulus intensity than normal. As a non-specific increase in membrane excitability seems to be associated with an influx of calcium through voltage-sensitive calcium channels, calcium antagonists, together with agents specifically blocking relevant transmitter substances, would offer an effective therapy against the unstable bladder.

Extracellular calcium dependence of the neurotensin-induced relaxation of intestinal smooth muscles: Studies with calcium channel blockers and BAY K-8644

To investigate whether the neurotensin-induced relaxation of the rat duodenum and ileum is dependent on the external concentration of calcium, the effect of the neuropeptide was studied in isolated intestinal segments superfused with Tyrode solution containing no calcium, 1 or 2.5 mM Ca 2+. The neurotensin-induced intestinal relaxation was reduced when the extracellular calcium concentration was lowered. In addition, the inhibitory effect of neurotensin was cancelled when the tissues were incubated in the presence of diltiazem, methoxyverapamil or nifedipine. BAY K-8644, a structural analog of nifedipine that functions as an agonist of the calcium channel, potentiated the neurotensin-induced smooth muscle relaxation. The facilitatory effect of BAY K-8644 was antagonized by nifedipine, indicating competition between the 2 dihydropyridines. Apamin, the K + channel blocker, antagonized the neurotensin-induced visceral relaxation, displacing the concentration-response curve of the peptide to the right. Furthermore, apamin also blocked the effect of neurotensin when the neuropeptide was assayed in the presence of BAY K-8644. It is concluded that the smooth muscle relaxation induced by neurotensin is dependent on external calcium, suggesting that the activation of the neuropeptide receptor causes an influx of calcium which leads to the opening of K + channels before smooth muscle relaxation is triggered.