Effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum

The effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum were investigated. The resting membrane potential of the duodenal smooth muscle cells was reduced by neurotensin at the concentration of 3 X 10(-8) M or more. The amplitude of the non-adrenergic inhibitory potentials was decreased by neurotensin (1-3 X 10(-8) M). In the Ca2+-free solution, the amplitude of the inhibitory potentials was also decreased. The increased amplitude of the non-adrenergic inhibitory potentials evoked in the high calcium solution (Ca2+, 5 mM) was decreased by neurotensin (10(-8) M). Neither atropine (1.4 X 10(-6) M) nor propranolol (3.4 X 10(-6) M) blocked the inhibitory action of neurotensin (10(-8) M) on the inhibitory potential. The frequency of the spontaneous action potentials of the duodenal smooth muscle cells was strongly increased with accompanying depolarization by neurotensin 8 X 10(-8) M. The tonic contraction of the duodenal smooth muscles was produced by neurotensin (3-8 X 10(-8) M). The results obtained suggest that neurotensin relates to the non-adrenergic inhibitory pathway in the control mechanism on intestinal motility.     

Effects of mechanical stretch on the membrane potential of guinea pig ventricular muscles

We studied the effect of stretch on the membrane potentials and ultrastructure of isolated ventricular papillary muscles of guinea pigs. The muscles were stimulated at 0.5 Hz and stretched stepwise from slack length (90% of Lmax) to 100% (mild stretch), 110-120% (moderate stretch), and 130-140% of Lmax (severe stretch), under microscopic control. In control Tyrode solution (K+ = 5.4 mM, Ca2+ = 1.8 mM, Mg2+ = 0.5 mM), the mild to moderate stretch significantly depolarized the resting potential (RP) by about 6 mV as compared to that in slack length, whereas the severe stretch hyperpolarized the membrane by about 5 mV. The latter finding was new and was focused on in later experiments. Both the hyperpolarization and depolarization became more marked when [K+]o was decreased to 1.35-2.7 mM, and became less with elevated [K+]o to 10.8-21.6 mM, thereby suggesting the participation of altered K+ conductance (gK) with these changes in the RP. Perfusion with low [Ca2+]o (0.45 mM) enhanced the depolarization but eliminated the hyperpolarization; high [Ca2+]o (7.2 mM) inhibited the depolarization without effect on the hyperpolarization. D-600 (1 microM), caffeine (10 mM), and ryanodine (1 microM), all of which may produce decreases in [Ca2+]i, abolished the hyperpolarization with inconsistent effects on the depolarization. Moderate to severe stretches decreased the maximum rate of rise of action potential (Vmax), by shifting the Vmax-RP relationship toward hyperpolarizing direction. The shift could be reversed partially after increasing [Mg2+]o to 8.0 mM. Electron microscopic examination revealed that the sarcoplasmic reticulum (SR) remained intact with mild to moderate stretches with significant lengthening of sarcomere length, while with a severe stretch, the SR showed a structural disarrangement with a non-uniform lengthening of sarcomere length. Our observations suggest that stretch-induced hyperpolarization is probably mediated by the increase in gK, presumably secondary to the increase in [Ca2+]i. Ca2+ may be released from the SR upon mechanical stretch of the organelle.     

Somatostatin inhibition of glucose-induced electrical activity in cultured rat islet cells.

Electrophysiological studies of rat islet cells in monolayer culture were undertaken to determine the role of transmembranous ionic fluxes in the inhibitory action of somatostatin on insulin release. In the presence of somatotropin release inhibiting factor (SRIF) (2.5 nM), hyperpolarization occured with or without glucose (16.6 mM) in the medium. SRIF also inhibited the incidence of glucose-induced spike activity. The inhibitory action of SRIF occurred within 5 min and was readily reversible. An increase in extracellular K+ (5-13 mM) or Ca2+ (2.3-4.6 mM) prevented SRIF inhibition of glucose-induced electrical activity. The secretory response of cultured islets to glucose (16.6 mM) was completely inhibited by SRIF (2.5 nM). The presence of high [Ca2+]o or [k+]o enhanced insulin release in the presence of SRIF and glucose. Although phentolamine (5.0 microgram/ml) did not block the inhibition of glucose-induced electrical responses by SRIF, it prevented the inhibitory action of epinephrine (0.2 microgram/ml). It is concluded that the primary action of SRIF is to alter transmembranous cationic fluxes, as manifested by hyperpolarization and a decrease in the incidence of spike activity, which may prevent glucose from eliciting a normal secretory response.     

Alpha-adrenoceptor agonists produce Ca2+ oscillations in isolated rat aorta: role of protein kinase C.

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) in responses to norepinephrine (NE) and selective alpha2-adrenoceptor agonist, UK14,304 of the endothelium-denuded rat aorta loaded with fura PE-3. NE (3 x 10(-8) M) evoked a rapid increase in [Ca2+]i followed by slight decreasing to a steady state level and produced a contraction. After the NE-induced increase in [Ca2+]i had reached a maximum, the [Ca2+]i showed persistent oscillations. The Ca2+ oscillations were superimposed on the sustained increase in [Ca2+]i. UK14,304 (3 x 10(-6) M) also evoked an increase in [Ca2+]i and produced a contraction. However, the UK14,304-induced effect on [Ca2+]i was characterized by pronounced oscillations, and the amplitude of the sustained increase in [Ca2+]i was less than that seen with NE. Protein kinase C inhibitor, Ro31-8220 (3 x 10(-6) M) and verapamil (10(-5) M) abolished both NE and UK14,304-evoked Ca2+ oscillations. UK14,304-induced contractions were also strongly inhibited by Ro31-8220 and verapamil. However, NE induced contractions were partly inhibited by these inhibitors. The sustained increases in [Ca2+]i evoked NE and UK14,304 were not significantly inhibited by Ro31-8220 and verapamil. These results suggest that NE and UK14,304 produce Ca2+ oscillations during sustained contractions in rat aorta. The alpha2 adrenoceptor agonist, UK14,304-induced sustained contraction and Ca2+ oscillations may be due to PKC activation and opening of voltage-dependent L type Ca2+ channels.     

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea-pig taenia coli.2. Replacement of external NaCl with sucrose (leaving 10 mM-Na in the buffer) caused hyperpolarization and stopped spontaneous activity within 10 min. Spikes could, however, be evoked for 2-3 hr. The amplitude, the overshoot and rate of rise of the spike were increased.3. In 10 mM-[Na](o) the intracellular Na concentration was reduced from 35 to 24 mM, shifting the Na-equilibrium potential from +34 to -22 mV.4. Excess Ca (12.5 mM) caused hyperpolarization and increased membrane conductance. The amplitude and the rate of rise of the spike were increased, the threshold was raised and the latency of the spike evoked by threshold stimulation became shorter.5. The effect of reducing the external Ca concentration depended on the Na concentration present, being greater with higher external [Na](o). When the membrane was depolarized and spikes deteriorated in low Ca (0.2-0.5 mM) reduction of Na to 10 mM caused repolarization and recovery of the action potential.6. Mn (0.5-1.0 mM) blocked spontaneous spike discharge after 20 min. Higher concentrations (more than 2.0 mM) were required to block the evoked action potential.7. The results indicate that the smooth muscle spike in taenia is due to Ca-entry and that Na influences spike activity indirectly by competing with Ca in controlling the membrane potential.     

Effects of neurotensin on electrical and mechanical properties of smooth muscles in longitudinal and circular layers of the guinea-pig ileum

Effects of neurotensin (NT) on the electrical and mechanical activities of longitudinal and circular muscles of the guinea-pig ileum were investigated using the micro-electrode and isometric tension recording methods. In longitudinal muscles, the resting membrane potential was not affected by NT (0.1–30 nmol/l), but NT did provoke the contraction when applied in concentrations over 1 nmol/l. TTX (0.1 mol/l) neither modified the resting membrane potential nor the contraction evoked by NT, under condition of pretreatment with - and -adrenoceptor blockers. In circular muscles, NT (over 0.1 nmol/l) consistently hyperpolarized the membrane and increased the ionic conductance. The hyperpolarization appeared with a transient hyperpolarization, which gradually declined with a long time course. Using apamin and various concentrations of Ca, the NT-induced hyperpolarization was classified into two subtypes; fast and slow. The former was composed of maximum hyperpolarization due to activations of the Ca independent K channel, and the latter was composed of late hyperpolarization, due to activations of the Ca dependent K channel. During the NT-induced hyperpolarization in circular muscles, the amplitude of non-adrenergic, noncholinergic inhibitory junction potential (i.j.p.) evoked by field stimulation was reduced. This reduction induced by 0.5 nmol/l NT was mainly due to hyperpolarization of the membrane, and that observed in a high concentration of NT (3 nmol/l) was directly involved in ionic mechanisms contributing to the generation of i.j.p. In circular muscles, NT (over 3 nmol/l) did relax the tissue pre-contracted with 17.8 mmol/l K, but NT (below 30 nmol/l) did not relax the tissue pre-contracted by 39.6 mmol/l K. The relaxation of the pre-contracted tissue induced by NT depended on the hyperpolarization induced by NT. The results indicate that NT has direct actions on muscle cells of both longitudinal and circular layers of the ileum. In longitudinal muscles, NT has no effect on the membrane potential but does produce contraction, while in circular muscles, NT activates the Ca independent and Ca dependent K channels and relaxes the tissue, by hyperpolarizing the membrane. The membrane response evoked by NT differs from that induced by the activation of non-adrenergic, non-cholinergic nerves.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Comparison of Mg, Mn, and Co ions affecting the beta-adrenoceptor-mediated membrane response in the guinea-pig taenia caeci

Electrical activity was recorded intracellularly from the muscle cell of guinea-pig taenia caeci in Locke solution. Membrane potential was -46.4 mV, and spike potentials were discharged spontaneously. Isoprenaline (3 microM) hyperpolarized the membrane and suppressed the spike discharge. The hyperpolarization by isoprenaline was increased at low K (2 mM), while decreased at high K (11.8, 29.5, 59 mM). The hyperpolarization by isoprenaline was potentiated in the presence of external Mg ions, depending on the concentration of Mg (0-9.6 mM). Forskolin (3 microM) and papaverine (30 microM) hyperpolarized the membrane; the effects were augmented by 1.2 mM Mg. The hyperpolarization in response to 3 microM isoprenaline or 100 microM papaverine was inhibited by Mn, Co, and low Ca (1 mM) whereas it was not affected by high Ca (7.5 mM). Verapamil (0.5, 2 microM) had no influence of the hyperpolarization caused by isoprenaline. It was discussed that extracellular and/or intracellular Mg and Ca ions played important roles in the beta-adrenoceptor-mediated action on the smooth muscle membrane of taenia caeci.     

Effects of enkephalin and endorphin on the inhibitory junction potentials in the duodenal smooth muscle cells of the guinea-pig

Inhibitory junction potentials (i.j.p.s) evoked by field stimulation were recorded from the smooth muscle cells of the guinea-pig duodenum intracellularly. The membrane potential was -54.3 mV. The parameters of the i.j.p. were as follows: latency, 71 msec; time to peak, 146 msec; amplitude, 15.5 mV; rate of hyperpolarization, 107 mV/sec; and half decay time of the i.j.p., 193 msec. Met-enkephalin (10(-7)-10(-6) M) had no effect on the membrane potential and the i.j.p. The membrane potential was decreased by beta-endorphin (1.7 X 10(-7)-6.8 X 10(-7) M). Increase in the latency and the time to peak and decrease in the amplitude and the rate of hyperpolarization of the i.j.p. were observed for beta-endorphin. "Spontaneous" excitatory junction potentials (e.j.p.s) were generated by beta-endorphin. Naloxone (3.1 X 10(-6)-3.1 X 10(-4) M) hyperpolarized the membrane of the muscle cells. At high concentrations of naloxone (3.1 X 10(-4) and 3.1 X 10(-3) M), inhibition of the i.j.p. was observed. Levallorphan (2.3 X 10(-4) M) prolonged the latency and the time to peak and reduced the amplitude of the i.j.p. The membrane potential was slightly decreased by levallorphan. "Spontaneous" e.j.p.s were generated by levallorphan in a certain population of the cells. It is concluded that Met-enkephalin does not contribute to the non-adrenergic inhibitory transmission and that beta-endorphin acts as a modulator in the control mechanism of the intestinal motility. The effects of naloxone and levallorphan on the i.j.p. are discussed.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Penetration-induced hyperpolarization as evidence for Ca2+ activation of K+ conductance in isolated smooth muscle cells

Single smooth muscle cells, freshly isolated by enzymatic digestion of the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using standard electrophysiological techniques. Following penetration with a microelectrode, a hyperpolarization lasting many seconds occurred before the membrane depolarized to a steady-state level. The following lines of evidence indicate that the penetration-induced hyperpolarization results from an increase in K+ conductance caused by Ca2+ that enters the cell at the time of penetration: 1) The cell contracted at the time of penetration indicating that [Ca2+]i was elevated even though no action potential had occurred; the cell subsequently relaxed. 2) The input resistance was much lower during the hyperpolarization than during the steady-state resting potential. In the steady state all cells displayed outward-going rectification. 3) At constant [Ca2+]0, the amplitude of the hyperpolarization varied with log[K+]0 (1.3-56 mM) to a much greater degree than did the steady-state potential. Tetraethylammonium chloride (TEA) (18.2 mM) reduced the hyperpolarization. 4) At constant [K+]0, the amplitude of the hyperpolarization increased as the [Ca2+]0 was raised (1.8-52.1 mM). 5) With [Ca2+]0 low (less than or equal to 0.16 mM), the hyperpolarization was almost completely abolished in the presence of a high concentration of Ba2+ (80 mM) or Mn2+ (79.2 mM); this was not the case with Sr2+.     

Divergent action of verapamil perfused in two hypothalamic areas on body temperature of the cat

Guide cannulae for push-pull perfusion were bilaterally implanted stereotaxically within the anterior hypothalamic, preoptic area (AH/POA) and posterior hypothalamus (PH) of the cat. Catecholamine-reactive sites were identified within AH/POA in which a microinjection of norepinephrine (NE) (5.0 micrograms) evoked a characteristic, transient hypothermia. Similarly the cation-reactive region within the PH was identified in which excess Ca2+ (25 mM) also evoked a hypothermic response. When verapamil was perfused at a rate of 25.0 microliters/min in a concentration of 0.4 or 2.0 micrograms/microliter within AH/POA at a NE-sensitive site, a concentration-dependent decline in the core temperature of the cat occurred. Conversely, verapamil perfused in the same manner with a Ca2+-reactive site caused an intense rise in the cat's body temperature which also was concentration dependent. These results show that the localized blockade of slow Ca2+ channels exerts direct, differential physiological effects within central nervous system tissue. In this case, verapamil mimics noradrenergic effects within the AH/POA; however, the hyperthermic response following Ca2+ channel blockade within tissue of the PH resembled that produced by ethyleneglycoltetraacetic acid or Na ions.     

Reversal of alpha 1-receptor mediated relaxation in intestinal smooth muscle

The alpha 1-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba2+ concentration was increased to 10-15 mM the response to phenylephrine was a contraction, and at Ba2+ concentrations in between the high and low range this response was biphasic--a relaxation followed by a contractile phase. The alpha 2-receptor agonist clonidine did not affect the tone of the Ba2+ contracted preparation. When the muscle preparation was contracted by Sr2+ (1-20 mM) in the presence of Ca2+ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca2+, 5 mM Ba2+ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca2+ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca2+ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba2+ and 2.5 mM Ca2+ could be reverted to the normal relaxation by the addition of 20 mM Mg2+. Increasing the K+ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba2+ could reverse the response of alpha 1 receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca2+.     

Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.     

An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction

The hyperpolarization produced by the application of 10^?5 M d-tubocurarine to the end-plate of mouse diaphragm-phrenic nerve preparation pretreated with an irreversible anticholinesterase was studied. This hyperpolarization has been attributed to a non-quantal release of acetylcholine by the nerve terminals. The usual hyperpolarization effect of about 10 mV was mimicked by adding 10^?7 M acetylcholine to the bath which contained 15mM Ca²⁺ to block the non-quantal release. The hyperpolarization effect was maximal (9.2 ± 0.8mV) in saline solution with the normal concentrations of Ca²⁺ (2mM) and K⁺ (5mM). Reducing [Ca²⁺] in the bath decreased the hyperpolarization effect. In Ca-free solution with 4.10^?4 M ethleneglycoltetra-acetate the hyperpolarization effect was3.8 ± 0.6mV. An increase of [Ca²⁺] also reduced the hyperpolarization effect and it was absent in 15mM of Ca²⁺. When the external concentration of Ca²⁺ was kept constant (2mM), both decreasing and increasing K⁺ concentration from 5 mM diminished the hyperpolarization effect. However, the decrease at elevated K⁺ concentrations was due to a reduction of membrane resistance and when the hyperpolarization effect was corrected for the change in size of the miniature endplate potential, it was unchanged in solutions with increased [K⁺] up to 13 mM. No traces of non-quantal release were observed during repetitive stimulation of the phrenic nerve in a medium containing 0.3 mM Ca²⁺. The hyperpolarization effect was not found in denervated muscles that lacked signs of neuromuscular transmission. The hyperpolarization effect was blocked by botulinum toxin while some evoked release of ACh was present. The time required for the block by 1 μg/ml toxin was shorter than for 0.25 μg/ml. The application of α-bungarotoxin produced a hyperpolarization of the membrane in the junctional region. The addition of d-tubocurarine failed to produce the hyperpolarization effect. The hyperpolarization effect was also absent when muscles were incubated with the desensitization potentiating drug, SKF-525A. The hyperpolarization effect was unchanged by replacement of Na⁺ with guanidine and was absent following the replacement of Na⁺ with arginine.The data suggest the channel responsible for the hyperpolarization effect is the same as that giving the endplate potential. The results are in agreement with the hypothesis that the hyperpolarization effect reflects a sustained non-quantal release of acetylcholine from the nerve terminal into the synaptic cleft.     

Effects of ionic calcium on the responses of canine testicular polymodal receptors to algesic substances

1. To explore possible mechanisms of the responses to algesic substances (bradykinin, hypertonic saline, and high K+ solution) of polymodal receptors in the canine testis, the Ca2+ concentration was varied in vitro. 2. After 1 min in Ca2+-free media, the responses to both high K+ solution (60 mM K+) and hypertonic saline (0.6 M Na+) were significantly augmented and tended to increase further with time; return to normal Ca2+ concentration quickly reversed these changes. These augmenting effects were blocked by the substitution of Mg2+ for Ca2+. The excitation produced by 60 mM K+ was decreased by increasing Ca2+ in a concentration-dependent fashion. 3. Reducing the bath concentration of K+ decreased responses evoked by 9 X 10(-8) M bradykinin (BK), whereas increased K+ concentration had the opposite effect. 4. The excitatory effects of BK were significantly suppressed in extracellular Ca2+-free condition. The suppression was not affected by the addition of Mg2+. Prostaglandin E2, which has been known to be released by BK and to augment the BK response, failed to restore the suppressed response by either preapplication (2.8 X 10(-7) M) or simultaneous application in high concentration (1.4 X 10(-5) M). 5. On the basis of these observations, it was postulated that Ca2+ concentration-dependent changes of the responses to 60 mM K+ and 0.6 M Na+ results from Ca2+-dependent "membrane surface potential" changes. The suppressed response to BK by Ca2+ depletion may be explained by the intervention of Ca2+-dependent processes other than PG production.     

Effects of external calcium reduction on biphasic potassium contractures and action of divalent cations on the calcium reduction in frog single twitch muscle fibers

The effects of external Ca2+ reduction on the biphasic potassium (K) contractures and the action of divalent cations under conditions of the Ca2+ reduction were examined in detail, using frog single twitch muscle fibers. The peak tension of the initial component of 80 mM K+ contractures was greatly potentiated by exposing the fiber to low Ca2+ solution for 30 sec, and the degree of this potentiation was decreased with increasing the exposing time. In contrast, the peak tension of the secondary component was rapidly inhibited by Ca2+ reduction. The potentiation of the initial component was removed by 3 mM Mg2+ or 0.5 mM Ni2+. The inhibition of the secondary component, especially the shortening of its time course, was reversed partially by 3 mM Mg2+ and almost completely by 0.5 mM Ni2+. The difference between the inhibitory effect of 10 mM Mg2+ or 1-3 mM Ni2+ on the initial component and that on the secondary component was also demonstrated. The tension development of the secondary component was completely inhibited by external Ca2+ reduction for 20 min, but it was observed in the presence of 3 mM Mg2+, although its time course was shorter. These results indicate that the actions of Mg2+ and Ni2+ on the initial component differ from those on the secondary component and suggest the possible mechanisms of the actions of these divalent cations on the biphasic K contractures.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine.

Superfusion of the isolated sympathetic ganglion of the bullfrog with a caffeine-containing (1-6 mM) solution caused in many cells an initial slow hyperpolarization which was followed by a subliminal depolarization interruped by rhythmic hyperpolarizations. A hyperpolarization, similar to one of the rhythmic hyperpolarizations, could be triggered by an action potential in the presence of caffeine. The action potential itself was not markedly affected by caffeine except for its afterhyperpolarization which was prolonged. All these caffeine-induced hyperpolarizations were associated with a marked reduction of the membrane resistance, their amplitude was increased in a K+-free solution and decreased in a high-K+ solution, and their polarity was reversed at the same level at which the afterhyperpolarization was also inverted. This reversal level was not altered by omission of Na+ or C1- from the external medium. These hyperpolarizations were reversibly abolished by depletion of external Ca2+ or replacement of external Ca2+ by Mg2+. Excess of external Ca2+ caused a shortening of the interval between rhythmic hyperpolarizations. Furthermore, iontophoretic injection of EDTA into the cytoplasm markedly depressed the initial caffeine hyperpolarizatin and abolished both the rhythmic and evoked caffeine hyperpolarizations. The caffeine-induced depolarization was not affected by omission of external Cl-. It was decreased in a Na+-free medium, but completely eliminated by omission of both Na+ and Ca2+ from the external medium. Tetrodotoxin did not impair the production of the initial and the rhythmic hyperpolarizations. A strong depolarizing pulse could evoke a typical hyperpolarizing response in the presence of this compound. Dibutyryl cyclic AMP, d-tubocurarine, atropine, and phenoxybenzamine were without effect on the caffeine-induced hyperpolarizations and depolarization. It was concluded that each caffeine-induced hyperpolarization is the result of an increased K+ permeability, which is probably caused by a rise in the internal Ca2+ concentration. It was also concluded that the caffeine-induced depolarization is due to an increased membrane permeability to Ca2+ and Na+.     

Interaction between secretin and cholecystokinin-octapeptide in the exocrine rat pancreas in vivo and in vitro.

This study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2+ and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10(-8) mol kg-1 h-1) or staurosporine (10(-8) mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10(-11) M) or secretin (10(-11) M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10(-6) M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10(-11) M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10(-11) M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK8. In superfused pancreatic segments CCK8 (10(-11) M) evoked a net efflux of Mg2+ whereas secretin (10(-11) M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.