Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro

Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX1R) and orexin 2 (OX2R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca2+ concentration ([Ca2+]i) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10(-8) to 10(-6)M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10(-7)M. When both ORX-A and ORX-B were applied simultaneously at 10(-7)M, the increase in firing rate was almost equal to that of ORX-B at 10(-7)M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX1R receptors. The excitatory effect of ORX-B was seen in low Ca2+ and high Mg2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca2+ and high Mg2+ ACSF than in normal ACSF. [Ca2+]i imaging studies demonstrated that [Ca2+]i was increased in about 50% of the PVT neurons by both 10(-7)M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca2+]i induced by ORX-B was abolished in Ca2+-free ACSF, suggesting that ORX-B does not release Ca2+ from intracellular Ca2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca2+-free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca2+-free and TTX-containing Ca2+-free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about -97 mV, and an increase of extracellular K+ concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K+ channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX2R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors.     

Electrophysiological effects of orexin-B and dopamine on rat nucleus accumbens shell neurons in vitro

Orexin (ORX) plays a critical role in reward-seeking behavior for natural rewards and drugs of abuse. The mesolimbic dopamine (DA) pathway that projects into the nucleus accumbens (NAc) from the ventral tegmental area is deeply involved in the neural mechanisms underlying reward, drug abuse and motivation. A recent study demonstrated that ORX-immunopositive fibers densely project into the shell of the NAc (NAcSh), suggesting that the NAcSh might be a site of the interaction between the ORXergic and DAergic systems for reward-seeking behavior. Therefore, the electrophysiological effects of ORX-B and DA on NAcSh neurons were examined extracellularly in rat brain slice preparations. ORX-B excited approximately 78% of neurons tested and inhibited 4%, whereas DA excited 50% and inhibited 22% of NAcSh neurons. These excitations and inhibitions persisted during synaptic blockade in a low-Ca²⁺/high-Mg²⁺ solution. DA-induced excitation was attenuated by SCH23390 or sulpiride, whereas DA-induced inhibition was suppressed by sulpiride. Of the neurons that were excited by ORX-B, 71% and 18% were excited and inhibited by DA, respectively. In 63% of neurons that were excited by ORX-B, the simultaneous application of ORX-B and DA increased the firing rate to two times greater than ORX-B alone, whereas, the simultaneous application significantly decreased the neuronal firing rate by 73% in the remaining 37% compared to ORX-B. These results suggest that an interaction between the ORXergic and DAergic systems occurs in the NAcSh and that the NAcSh is involved in the neural mechanisms in which ORX participates in the regulation of reward-seeking behavior.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Histamine-induced depolarization: ionic mechanisms and role in sustained contraction of rabbit cerebral arteries

The role of membrane depolarization in the histamine-induced contraction of the rabbit middle cerebral artery was examined by simultaneous measurements of membrane potential and isometric force. Histamine (1-100 microM) induced a concentration-dependent sustained contraction associated with sustained depolarization. Action potentials were observed during depolarization caused by histamine but not by high-K(+) solution. K(+)-induced contraction was much smaller than sustained contraction associated with the same depolarization caused by histamine. Nifedipine attenuates histamine-induced sustained contraction by 80%, with no effect on depolarization. Inhibition of nonselective cation channels with Co(2+) (100-200 microM) reversed the histamine-induced depolarization and relaxed the arteries but induced only a minor change in K(+)-induced contraction. In the presence of Co(2+) and in low-Na(+) solution, histamine-evoked depolarization and contraction were transient. We conclude that nonselective cation channels contribute to histamine-induced sustained depolarization, which stimulates Ca(2+) influx through voltage-dependent Ca(2+) channels participating in contraction. The histamine-induced depolarization, although an important and necessary mechanism, cannot fully account for sustained contraction, which may be due in part to augmentation of currents through voltage-dependent Ca(2+) channels and Ca(2+) sensitization of the contractile process.     

Hypocretin/orexin suppresses corticotroph responsiveness in vitro

The hypocretin/orexins (Hcrts/ORXs) are peptides produced in neurons in the lateral hypothalamic area that project to neuroendocrine centers in the hypothalamus. Hcrt/ORX receptors are present in the hypothalamus and anterior pituitary gland. We examined the possibility that the Hcrts/ORXs, which we have demonstrated previously to act in the brain to stimulate sympathetic function, could alter stress hormone secretion by a direct pituitary action. In vitro studies revealed a dose-related inhibitory effect of the Hcrts/ORXs on corticotropin-releasing hormone-stimulated ACTH secretion that appeared to be mediated via the orexin-1 receptor and to be expressed at doses (threshold dose 1 nM orexin A) similar to the affinity constant for the receptor. The effect was not due to abrogation of the cAMP response of the corticotroph to corticotropin-releasing hormone and was not pertussis toxin sensitive, suggesting a non-G(i)-mediated mechanism. Instead, a G(q)-mediated signaling mechanism was indicated by the ability of protein kinase C blockade with calphostin C to reverse the inhibitory action of orexin A. Orexin A and orexin B did not significantly alter basal ACTH secretion in vitro and did not alter basal or releasing factor-stimulated secretion of luteinizing hormone, prolactin, thyroid-stimulating hormone or growth hormone from cells harvested from male or random-cycle female donors. Our data suggest a direct, pituitary action of the Hcrts/ORXs to modulate the endocrine response to stress and identify the potential cellular mechanism of a unique biological action of the peptides in the anterior pituitary gland.     

Prokineticin 2 influences subfornical organ neurons through regulation of MAP kinase and the modulation of sodium channels

Prokineticin 2 (PK2) is a neuropeptide that acts as a signaling molecule regulating circadian rhythms in mammals. We have previously reported PK2 actions on subfornical organ (SFO) neurons, identifying this circumventricular organ as a target at which PK2 acts to influence autonomic control (Cottrell GT, and Ferguson AV. J. Neurosci. 24: 2375-2379, 2004). In this study, we have examined the cellular mechanisms by which PK2 increases the excitability of SFO neurons. Whole cell patch recordings from dissociated rat SFO neurons demonstrated that the mitogen-activated protein (MAP) kinase inhibitor PD-98059 prevented PK2-induced depolarization and decreases in delayed rectifier K(+) current. PK2 also increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in 39% of dissociated SFO neurons (mean increase = 20.8 +/- 5.5%), effects that were maintained in the presence of thapsigargin but abolished by both nifedipine, or the absence of extracellular Ca(2+), suggesting that PK2-induced [Ca(2+)](i) transients resulted from Ca(2+) entry through voltage-gated Ca(2+) channels. Voltage-clamp recordings showed that PK2 was without effects on Ca(2+) currents evoked by voltage ramps, suggesting that PK2-induced Ca(2+) influx was secondary to PK2-induced increases in action potential frequency, an hypothesis supported by data showing that tetrodotoxin abolished effects of PK2 on [Ca(2+)](i). These observations suggested PK2 modulation of voltage-gated Na(+) currents, a possibility confirmed by voltage-clamp experiments showing that PK2 increased the amplitude of both transient and persistent Na(+) currents in 29% of SFO neurons (by 34 and 38%, respectively). These data indicate that PK2 influences SFO neurons through the activation of a MAP kinase cascade, which, in turn, modulates Na(+) and K(+) conductances.     

Regulation of neuronal K(+) currents by target-derived factors: opposing actions of two different isoforms of TGFbeta.

The developmental expression of macroscopic Ca(2+)-activated K(+) currents in chick ciliary ganglion neurons is dependent on an avian ortholog of TGFbeta1, known as TGFbeta4, secreted from target tissues in the eye. Here we report that a different isoform, TGFbeta3, is also expressed in a target tissue of ciliary ganglion neurons. Application of TGFbeta3 inhibits the functional expression of whole-cell Ca(2+)-activated K(+) currents evoked by 12 hour treatment with either TGFbeta1 or beta-neuregulin-1 in ciliary ganglion neurons developing in vitro. TGFbeta3 had no effect on voltage-activated Ca(2+) currents. A neutralizing antiserum specific for TGFbeta3 potentiates stimulation of Ca(2+)-activated K(+) currents evoked by a target tissue (iris) extract in cultured ciliary ganglion neurons, indicating that TGFbeta3 is an inhibitory component of these extracts. Intraocular injection of TGFbeta3 causes a modest but significant inhibition of the expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo. Further, intraocular injection of a TGFbeta3-neutralizing antiserum stimulates expression of Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo, indicating that endogenous TGFbeta3 regulates the functional expression of this current. The normal developmental expression of functional Ca(2+)-activated K(+) currents in ciliary ganglion neurons developing in vivo is therefore regulated by two different target-derived isoforms of TGFbeta, which produce opposing effects on the electrophysiological differentiation of these neurons.     

Ca(2+)-dependent desensitization of insulin secretion by strong potassium depolarization

Depolarization by a high K(+) concentration is a widely used experimental tool to stimulate insulin secretion. The effects occurring after the initial rise in secretion were investigated here. After the initial peak a fast decline occurred, which was followed by a slowly progressive decrease in secretion when a strong K(+) depolarization was used. At 40 mM KCl, but not at lower concentrations, the decrease continued when the glucose concentration was raised from 5 to 10 mM, suggesting an inhibitory effect of the K(+) depolarization. When tolbutamide was added instead of the glucose concentration being raised, a complete inhibition down to prestimulatory values was observed. Equimolar reduction of the NaCl concentration to preserve isoosmolarity enabled an increase in secretion in response to glucose. Unexpectedly, the same was true when the Na(+)-reduced media were made hyperosmolar by choline chloride or mannitol. The insulinotropic effect of tolbutamide was not rescued by the compensatory reduction of NaCl, suggesting a requirement for activated energy metabolism. These inhibitory effects could not be explained by a lack of depolarizing strength or by a diminished free cytosolic Ca(2+) concentration ([Ca(2+)](i)). Rather, the complexation of extracellular Ca(2+) concomitant with the K(+) depolarization markedly diminished [Ca(2+)](i) and attenuated the inhibitory action of 40 mM KCl. This suggests that a strong but not a moderate depolarization by K(+) induces a [Ca(2+)](i)-dependent, slowly progressive desensitization of the secretory machinery. In contrast, the decline immediately following the initial peak of secretion may result from the inactivation of voltage-dependent Ca(2+) channels.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.     

Actions of reactive oxygen species on AH/type 2 myenteric neurons in guinea pig distal colon

With conventional intracellular recording methods, we investigated the mechanism of actions of reactive oxygen species (ROS) derived from hypoxanthine and xanthine oxidase (HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons showed a transient membrane hyperpolarization(s) followed by a long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a late-onset membrane depolarization, respectively, and the remaining two neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca(2+)-dependent K(+) channels are the ionic mechanisms underlying the membrane hyperpolarization and depolarization, respectively. The effects of HX/XO on myenteric neurons were mimicked by application of caffeine or H(2)O(2). The results suggest that OH(.), but neither H(2)O(2) nor O(2)(.-), is responsible for HX/XO-induced responses. The intracellular Ca(2+) store may be the acting site of ROS in colonic AH/type 2 neurons.     

Dopaminergic modulation of axon initial segment calcium channels regulates action potential initiation

Action potentials initiate in the axon initial segment (AIS), a specialized compartment enriched with Na(+) and K(+) channels. Recently, we found that T- and R-type Ca(2+) channels are concentrated in the AIS, where they contribute to local subthreshold membrane depolarization and thereby influence action potential initiation. While periods of high-frequency activity can alter availability of AIS voltage-gated channels, mechanisms for long-term modulation of AIS channel function remain unknown. Here, we examined the regulatory pathways that control AIS Ca(2+) channel activity in brainstem interneurons. T-type Ca(2+) channels were downregulated by dopamine receptor activation acting via protein kinase C, which in turn reduced neuronal output. These effects occurred without altering AIS Na(+) or somatodendritic T-type channel activity and could be mediated by endogenous dopamine sources present in the auditory brainstem. This pathway represents a new mechanism to inhibit neurons by specifically regulating Ca(2+) channels directly involved in action potential initiation.     

Reduced functional expression of K(+) channels in vascular smooth muscle cells from rats made hypertensive with N{omega}-nitro-L-arginine

Smooth muscle membrane potential is determined, in part, by K(+) channels. In the companion paper to this article, we demonstrated that superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-l-arginine (l-NNA) are depolarized and express less K(+) channel protein compared with those from normotensive rats. In the present study, we used patch-clamp techniques to test the hypothesis that l-NNA-induced hypertension reduces the functional expression of K(+) channels in smooth muscle. In whole cell experiments using a Ca(2+)-free pipette solution, current at 0 mV, largely due to voltage-dependent K(+) (K(V)) channels, was reduced approximately 60% by hypertension (2.7 +/- 0.4 vs. 1.1 +/- 0.2 pA/pF). Current at +100 mV with 300 nM free Ca(2+), largely due to large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels, was reduced approximately 40% by hypertension (181 +/- 24 vs. 101 +/- 28 pA/pF). Current blocked by 3 mM 4-aminopyridine, an inhibitor of many K(V) channel types, was reduced approximately 50% by hypertension (1.0 +/- 0.4 vs. 0.5 +/- 0.2 pA/pF). Current blocked by 1 mM tetraethylammonium, an inhibitor of BK(Ca) channels, was reduced approximately 40% by hypertension (86 +/- 14 vs. 53 +/- 19 pA/pF). Differences in BK(Ca) current magnitude are not attributable to changes in single-channel conductance or Ca(2+)/voltage sensitivity. The data support the hypothesis that l-NNA-induced hypertension reduces K(+) current in vascular smooth muscle. Reduced molecular and functional expression of K(+) channels may partly explain the depolarization and augmented contractile sensitivity of smooth muscle from l-NNA-treated rats.     

Membrane potential modulates divalent cation entry in rat parotid acini

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

Vascular smooth muscle cell membrane depolarization after NOS inhibition hypertension

Nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-L-arginine (L-NNA) produces L-NNA hypertensive rats (LHR), which exhibit increased sensitivity to voltage-dependent Ca(2+) channel-mediated vasoconstriction. We hypothesized that enhanced contractile responsiveness after NOS inhibition is mediated by depolarization of membrane potential (E(m)) through attenuated K(+) channel conductance. E(m) measurements demonstrated that LHR vascular smooth muscle cells (VSMCs) are depolarized in open, nonpressurized (-44.5 +/- 1.0 mV in control vs. -36.8 +/- 0.8 mV in LHR) and pressurized mesenteric artery segments (-41.8 +/- 1.0 mV in control vs. -32.6 +/- 1.4 mV in LHR). Endothelium removal or exogenous L-NNA depolarized control VSMCs but not LHR VSMCs. Superfused L-arginine hyperpolarized VSMCs from both the control and LHR groups and reversed L-NNA-induced depolarization (-44.5 +/- 1.0 vs. -45.8 +/- 2.1 mV). A Ca(2+)-activated K(+) channel agonist, NS-1619 (10 microM), hyperpolarized both groups of arteries to a similar extent (from -50.8 +/- 1.0 to -62.5 +/- 1.2 mV in control and from -43.7 +/- 1.1 to -55.6 +/- 1.2 mV in LHR), although E(m) was still different in the presence of NS-1619. In addition, superfused iberiotoxin (50 nM) depolarized both groups similarly. Increasing the extracellular K(+) concentration from 1.2 to 45 mM depolarized E(m), as predicted by the Goldman-Hodgkin-Katz equation. These data support the hypothesis that loss of NO activation of K(+) channels contributes to VSMC depolarization in L-NNA-induced hypertension without a change in the number of functional large conductance Ca(2+)-activated K(+) channels.     

Role of potassium channels in catecholamine secretion in the rat adrenal gland

We elucidated the functional contribution of K(+) channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca(2+)-activated K(+) (SK(Ca))-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1, 1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 microM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 microM) and methacholine (10-300 microM). The putative large-conductance Ca(2+)-activated K(+) channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the K(A) channel blocker mast cell degranulating peptide (100-1000 nM) nor the K(V) channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SK(Ca) channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca(2+)-activated K(+) channels may control the secretion at the presynaptic site.