Omega-agatoxin-TK is a useful tool to study P-type Ca2+ channel-mediated changes in internal Ca2+ and glutamate release in depolarised brain nerve terminals

The present study shows that omega-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca(2+) channel blocker, omega-agatoxin-IVA in the venom, inhibits the high K(+) depolarisation-induced rise in internal Ca(2+) (Ca(i), as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC(50)'s of about 60nM. The maximal inhibition exerted by omega-agatoxin-TK in striatal synaptosomes (61 +/- 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of omega-agatoxin-TK-sensitive Ca(2+) channels in striatal than in hippocampal nerve endings. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1muM), inhibits part of the omega-agatoxin-TK-insensitive rise in Ca(i) induced by high K(+). In contrast to the inhibition exerted by omega-agatoxin-TK on the Ca(i) response to high K(+), omega-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Ca(i) and in internal Na(+) (Na(i), as determined with SBFI) induced by veratridine, indicating that the Ca(2+) influx activated by veratridine does not involve omega-agatoxin-TK-sensitive channels. High K(+) does not increase Na(i). In [(3)H]Glu preloaded hippocampal synaptosomes super-fused with low Na(+) Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [(3)H]Glu induced by high K(+) is absolutely dependent on the entrance of external Ca(2+). This exocytotic release of [(3)H]Glu attained in the absence of a chemical Na(+) gradient is inhibited with the same potency and efficacy by omega-agatoxin-TK and by omega-agatoxin-IVA, which is known to differ from omega-agatoxin-TK in its amino terminal moiety. These results indicate that omega-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca(2+) channel-mediated responses in cerebral nerve endings.     

Inhibitory effects of 1,4-DHP antagonists on synaptic GABA release modulated by BAY-K 8644 in mechanically dissociated rat substantia innominata

The effects of dihydropyridine (1,4-DHP) agonist and antagonists on miniature inhibitory postsynaptic currents (mIPSCs) were investigated in mechanically dissociated rat substantia innominata neurons attached to native GABAergic presynaptic nerve terminals, namely 'synaptic bouton preparation', using nystatin perforated patch recording mode under voltage-clamp conditions. BAY-K 8644 (BAY-K), an L-type Ca(2+) channel agonist, reversibly and concentration dependently facilitated the GABAergic mIPSC frequency without altering the distribution of current amplitudes. Removal of extracellular Ca(2+) completely suppressed the facilitatory effect of BAY-K on mIPSC frequency. The facilitatory effect of BAY-K on mIPSC frequency was maintained even in the presence of selective N-, P- and Q-type Ca(2+) channel antagonists, such as 3 x 10(-6) M omega-conotoxin-GVIA (omega-CgTX-GVIA), 3 x 10(-8) M omega-agatoxin-IVA (omega-AgTX-IVA) and 3 x 10(-6)M omega-conotoxin-MVIIC (omega-CmTX-MVIIC). However, nicardipine (3 x 10(-6) M) and nimodipine (3 x 10(-6) M), 1,4-DHP antagonists, significantly inhibited the mIPSC frequency enhanced by BAY-K by 37 +/- 5 and 42 +/- 6%, respectively. These results suggest the possible existence of L-type Ca(2+) channels in GABAergic presynaptic nerve terminals.     

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.     

Voltage-operated Ca(2+) channels involved in K(+)-evoked release of vasoactive intestinal polypeptide from the rat hypothalamus

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.     

Potentiation by stannous chloride of calcium entry into osteoblastic MC3T3-E1 cells through voltage-dependent L-type calcium channels

The present study was undertaken to confirm that L-type Ca(2+) channels are involved in Ca(2+) entry into osteoblastic MC3T3-E1 cells and to examine the effect of SnCl2, a Ca(2+)]-channel activator, on the intracellular Ca(2+)concentration ([Ca(2+)]i). High K(+)concentration-dependently raised the [Ca(2+)]i. All of the L-type Ca(2+)channel blockers used here, such as nifedipine, nicardipine, verapamil, and diltiazem, and CdCl2 (a non-selective blocker) inhibited the high K(+)-induced [Ca(2+)]i rise, but v-conotoxin GVIA (an N-type blocker) and NiCl2(a T-type blocker) had no effect. Application of SnCl2 alone did not change the [Ca(2+)]i. However, in the presence of high K(+), SnCl2 enhanced the high K(+)-induced [Ca(2+)]i rise, which was inhibited by Ca(2+)]-free medium or nifedipine. In the case where high K(+)was applied prior to SnCl2, SnCl2 alone raised the [Ca(2+)]i by itself. In conclusion, MC3T3-E1 cells possess the voltage-dependent L-type Ca(2+)] channels and SnCl2 facilitates the Ca(2+) entry through the L-type ones under the condition of the membrane depolarization. There is the possibility that Ca(2+) release from intracellular Ca(2+) stores is involved in the action of SnCl2.     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

Voltage-dependent calcium channels in the corpora allata of the adult male loreyi leafworm, Mythimna loreyi

In the corpora allata (CA) of the adult male loreyi leafworm, Mythimna loreyi, juvenile hormone acid (JHA) biosynthesis and release show a dose dependence on extracellular Ca(2+) concentration. Maxima are obtained with Ca(2+) concentrations of 2-10 mM, and synthesis and release are significantly inhibited under a Ca(2+)-free condition. The Ca(2+)-free inhibition of JHA release can be reversed by returning the glands to medium at 5 mM Ca(2+). The cytosolic free Ca(2+) concentration ([Ca(2+)](i)), which was measured with fura-2, in individual CA cells also shows a dose dependence on extracellular Ca(2+) concentration, with significant [Ca(2+)](i) depression being observed in the absence of extracellular Ca(2+).High K(+) significantly increases the JHA release and causes a transient [Ca(2+)](i) increase within seconds in CA cells. High-K(+)-stimulated JHA release is partially inhibited by the benzothiazepine (BTZ)-, dihydropyridine (DHP)- and phenylalkylamine (PAA)-sensitive L-type voltage-dependent calcium channel (VDCC) antagonists diltiazem, nifedipine and verapamil, respectively; by the N- and P/Q-type VDCC antagonist omega-conotoxin (omega-CgTx) MVIIC; and by the T-type VDCC antagonist amiloride. The N-type antagonist omega-CgTx GVIA is the most potent in inhibiting the high-K(+)-stimulated JHA release. No inhibitory effect is shown by the P-type antagonist omega-agatoxin TK (omega-Aga TK). The high-K(+)-induced transient [Ca(2+)](i) increase is largely inhibited by the L-type antagonists (diltiazem, nifedipine, verapamil), by the N- and P/Q-type antagonist omega-CgTx MVIIC and by the T-type antagonist amiloride, and is totally inhibited by the N-type antagonist omega-CgTx GVIA. No inhibitory effect is shown by the P-type antagonist omega-Aga TK.We hypothesize that L-type, N-type and T-type VDCCs may be involved to different degrees in the high-K(+)-stimulated JHA release and transient [Ca(2+)](i) increase in the individual CA cells of the adult male M. loreyi, and that the N-type VDCCs may play important roles in these cellular events.     

The involvement of voltage-operated calcium channels in somato-dendritic oxytocin release

Magnocellular neurons of the supraoptic nucleus (SON) secrete oxytocin and vasopressin from axon terminals in the neurohypophysis, but they also release large amounts of peptide from their somata and dendrites, and this can be regulated both by activity-dependent Ca(2+) influx and by mobilization of intracellular Ca(2+). This somato-dendritic release can also be primed by agents that mobilise intracellular Ca(2+), meaning that the extent to which it is activity-dependent, is physiologically labile. We investigated the role of different Ca(2+) channels in somato-dendritic release; blocking N-type channels reduced depolarisation-induced oxytocin release from SONs in vitro from adult and post-natal day 8 (PND-8) rats, blocking L-type only had effect in PND-8 rats, while blocking other channel types had no significant effect. When oxytocin release was primed by prior exposure to thapsigargin, both N- and L-type channel blockers reduced release, while P/Q and R-type blockers were ineffective. Using confocal microscopy, we found immunoreactivity for Ca(v)1.2 and 1.3 channel subunits (which both form L-type channels), 2.1 (P/Q type), 2.2 (N-type) and 2.3 (R-type) in the somata and dendrites of both oxytocin and vasopressin neurons, and the intensity of the immunofluorescence signal for different subunits differed between PND-8, adult and lactating rats. Using patch-clamp electrophysiology, the N-type Ca(2+) current density increased after thapsigargin treatment, but did not alter the voltage sensitivity of the channel. These results suggest that the expression, location or availability of N-type Ca(2+) channels is altered when required for high rates of somato-dendritic peptide release.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

Cerebral artery K(ATP)- and K(Ca)-channel activity and contractility: changes with development

The present study was designed to test the hypothesis that in cerebral arteries of the fetus, ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) channels (K(Ca)) play an important role in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) and that this differs significantly from that of the adult. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, simultaneously we measured norepinephrine (NE)-induced responses of vascular tension and [Ca(2+)](i) in the absence and presence of selective K(+)-channel openers/blockers. In fetal MCA, in a dose-dependent manner, both the K(ATP)-channel opener pinacidil and the K(Ca)-channel opener NS 1619 significantly inhibited NE-induced tension [negative logarithm of the half-maximal inhibitory concentration (pIC(50)) = 5.0 +/- 0.1 and 8.2 +/- 0.1, respectively], with a modest decrease of [Ca(2+)](i). In the adult MCA, in contrast, both pinacidil and NS 1619 produced a significant tension decrease (pIC(50) = 5.1 +/- 0.1 and 7.6 +/- 0.1, respectively) with no change in [Ca(2+)](i). In addition, the K(Ca)-channel blocker iberiotoxin (10(-7) to 10(-6) M) resulted in increased tension and [Ca(2+)](i) in both adult and fetal MCA, although the K(ATP)-channel blocker glibenclamide (10(-7) to 3 x 10(-5) M) failed to do so. Of interest, administration of 10(-7) M iberiotoxin totally eliminated vascular contraction and increase in [Ca(2+)](i) seen in response to 10(-5) M ryanodine. In precontracted fetal cerebral arteries, activation of the K(ATP) and K(Ca) channels significantly decreased both tension and [Ca(2+)](i), suggesting that both K(+) channels play an important role in regulating L-type channel Ca(2+) flux and therefore vascular tone in these vessels. In the adult, K(ATP) and the K(Ca) channels also appear to play an important role in this regard; however, in the adult vessel, activation of these channels with resultant vasorelaxation can occur with no significant change in [Ca(2+)](i). These channels show differing responses to inhibition, e.g., K(Ca)-channel inhibition, resulting in increased tension and [Ca(2+)](i), whereas K(ATP)-channel inhibition showed no such effect. In addition, the K(Ca) channel appears to be coupled to the sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma membrane K(+)-channel activity may account, in part, for the differences in the regulation of contractility of fetal and adult cerebral arteries.     

In eNOS knockout mice skeletal muscle arteriolar dilation to acetylcholine is mediated by EDHF

The mechanisms that account for acetylcholine (ACh)-induced responses of skeletal muscle arterioles of mice lacking endothelial nitric oxide (NO) synthase (eNOS-KO) were investigated. Isolated, cannulated, and pressurized arterioles of gracilis muscle from male eNOS-KO (74.1 +/- 2.3 microm) and wild-type (WT, 87.2 +/- 2.1 microm) mice developed spontaneous tone accounting for 63 and 61% of their passive diameter (116.8 +/- 3.4 vs. 143.2 +/- 2.8 microm, respectively) and dilated dose-dependently to ACh (10(-9)-10(-7) M). These dilations were significantly smaller in vessels of eNOS-KO compared with WT mice (29.2 +/- 2.0 microm vs. 46.3 +/- 2.1 microm, at maximum concentration) but responses to the NO donor, sodium nitrite (NaNO(2), 10(-6)-3 x 10(-5) M), were comparable in the vessels of the two strains. N(G)-nitro-L-arginine (L-NNA, 10(-4) M), an inhibitor of eNOS, inhibited ACh-induced dilations by 60-90% in arterioles of WT mice but did not affect responses in those of eNOS-KO mice. In arterioles of eNOS-KO mice, dilations to ACh were not affected by indomethacin but were essentially abolished by inhibitors of cytochrome P-450, clotrimazole (CTZ, 2 x 10(-6) M) or miconazole (MCZ, 2 x 10(-6) M), as well as by either high K(+) (40 mM) or iberiotoxin [10(-7) M, a blocker of Ca(2+)-dependent K(+) channels (K(Ca) channels)]. On the other hand, in WT arterioles CTZ or MCZ inhibited ACh-induced dilations only by approximately 10% and only in the presence of L-NNA. These results indicate that in arterioles of eNOS-KO mice, endothelium-derived hyperpolarizing factor (EDHF), synthesized via cytochrome P-450, accounts entirely for the mediation of ACh-induced dilation via an increase in K(Ca)-channel activity. In contrast, in arterioles of WT mice, endothelium-derived NO predominantly mediates ACh-induced dilation in which participation of EDHF becomes apparent only after inhibition of NO synthesis.     

Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals.

Multiple types of voltage-activated calcium (Ca(2+)) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca(2+) influx through N- and L- type voltage-activated Ca(2+) channels in sympathetic neurons to the depolarization-induced activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine (NE) synthesis, and the depolarization-induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both omega-conotoxin GVIA (1 microM), a specific blocker of N-type channels, and nimodipine (1 microM), a specific blocker of L-type Ca(2+) channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K(+), indicating that Ca(2+) influx through both types of channels contributes to enzyme activation. In contrast, K(+) stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by omega-conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K(+) stimulation of NE release from both ganglia and irises was also blocked completely when omega-conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca(2+) influx through N- and L-type Ca(2+) channels.     

NO(+) but not NO radical relaxes airway smooth muscle via cGMP-independent release of internal Ca(2+)

We compared the effects of two redox forms of nitric oxide, NO(+) [liberated by S-nitroso-N-acetyl-penicillamine (SNAP)] and NO. [liberated by 3-morpholinosydnonimine (SIN-1) in the presence of superoxide dismutase], on cytosolic concentration of Ca(2+) ([Ca(2+)](i); single cells) and tone (intact strips) obtained from human main stem bronchi and canine trachealis. SNAP evoked a rise in [Ca(2+)](i) that was unaffected by removing external Ca(2+) but was markedly reduced by depleting the internal Ca(2+) pool using cyclopiazonic acid (10(-5) M). Dithiothreitol (1 mM) also antagonized the Ca(2+) transient as well as the accompanying relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those to 60 mM KCl, suggesting the involvement of an electromechanical coupling mechanism rather than a direct effect on the contractile apparatus or on Ca(2+) channels. SNAP relaxations were sensitive to charybdotoxin (10(-7) M) or tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM). Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic monophosphate had any significant effect on resting [Ca(2+)](i), although both of these agents were able to completely reverse tone evoked by carbachol (10(-7) M). We conclude that NO(+) causes release of internal Ca(2+) in a cGMP-independent fashion, leading to activation of Ca(2+)-dependent K(+) channels and relaxation, whereas NO. relaxes the airways through a cGMP-dependent, Ca(2+)-independent pathway.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

EDHF,but not NO or prostaglandins,is critical to evoke a conducted dilation upon ACh in hamster arterioles

Vasomotor reactions upon focal stimulation of arterioles have been shown to be conducted along the vascular wall. Such a conduction, which is assumed to reflect the spread of electrical signals, may contribute to coordination of responses within a vascular segment. We aimed to identify which endothelial autacoid(s) act as mediators of the local and conducted dilator responses, respectively. To this end, arterioles in the hamster cremaster microcirculation were locally stimulated with endothelium-dependent [acetylcholine (ACh)] or endothelium-independent dilators [sodium nitroprusside (SNP)], and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.4 mm upstream at distant sites. Experiments were also performed after blockade of nitric oxide (NO) synthase, cyclooxygenase, P-450 monooxygenase, or K(+) channels. Dilations upon ACh (71 +/- 3%) were conducted rapidly (<1 s) to upstream sites (at 1.4 mm: 37 +/- 5%). Although the NO donor SNP induced a similar local dilation (71 +/- 7%), this response was not conducted. Maximal amplitudes of ACh-induced dilations were not attenuated after inhibition of NO synthase and cyclooxygenase at the local and remote sites. However, additional treatment with a P-450 monooxygenase blocker (sulfaphenazole) strongly attenuated the local response (from 62 +/- 9 to 17 +/- 5%) and abrogated dilations at distant sites (at 0.67 mm: from 23 +/- 4% to 4 +/- 3%). Likewise, 17-octadecynoic acid strongly attenuated local and remote responses. Blockers of Ca(2+)-dependent K(+) channels (charybdotoxin or iberiotoxin) attenuated dilations at the local and remote sites after focal application at the ACh stimulation site. In marked contrast, treatment of the upstream site with these blockers was without any effect. We conclude that upon local stimulation with ACh, a cytochrome P-450 monooxygenase product is generated that induces local dilation via the activation of Ca(2+)-dependent K(+) channels and initiates conduction of the dilation. In contrast to the local site, neither activation of these K(+) channels nor the synthesis of NO or prostaglandins is necessary to dilate the arterioles at remote, distant sites. This suggests that endothelium-derived hyperpolarizing factor serves as an important mediator to initiate conducted dilations and, by doing so, may act as a key player in the coordination of arteriolar behavior in the microcirculatory network.     

Cerebral artery sarcoplasmic reticulum Ca(2+) stores and contractility: changes with development

To test the hypothesis that sarcoplasmic reticulum (SR) Ca(2+) stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca(2+) stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10(-6) M), the NE-induced responses of tension and [Ca(2+)](i) were 37 +/- 5 and 47 +/- 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca(2+), adult MCA increases in tension and [Ca(2+)](i) were 32 +/- 5 and 13 +/- 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 x 10(-6) to 10(-5) M) resulted in increases in tension and [Ca(2+)](i) in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca(2+)](i) responses to RYN were essentially eliminated in the presence of zero extracellular Ca(2+). These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca(2+) stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca(2+) flux via plasma membrane L-type Ca(2+) channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca(2+)-activated K(+) channel and/or L-type Ca(2+) channel.     

Amlodipine inhibits thapsigargin-sensitive CA(2+) stores in thrombin-stimulated vascular smooth muscle cells

Ca(2+) channel blockers, such as amlodipine, inhibit vascular smooth muscle cell (VSMC) growth through interactions with targets other than L-type Ca(2+) channels. The effects of amlodipine on Ca(2+) movements in thrombin- and thapsigargin-stimulated VSMCs were therefore investigated by determining the variations of intracellular free Ca(2+) concentration in fura 2-loaded cultured VSMCs. Results indicated that 10-1,000 nM amlodipine inhibited 1) thrombin-induced Ca(2+) mobilization from a thapsigargin-sensitive pool and 2) thapsigargin-induced Ca(2+) responses, including Ca(2+) mobilization from internal stores and store-operated Ca(2+) entry. These effects of amlodipine do not involve L-type Ca(2+) channels and could not be reproduced with 100 nM isradipine, diltiazem, or verapamil. The inhibition by amlodipine of Ca(2+) mobilization appears therefore to be a specific property of the drug, in addition to its Ca(2+) channel-blocking property. It is suggested that amlodipine acts in this capacity by interacting with Ca(2+)-ATPases of the sarcoplasmic reticulum, thus modulating the enzyme activity. This mechanism might participate in the inhibitory effect of amlodipine on VSMC growth.