Kir3.1/3.2 encodes an I(KACh)-like current in gastrointestinal myocytes.
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Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K(+) channel subfamily encode G protein-gated inwardly rectifying K(+) channels (I(KACh)) in other tissues, including the heart and brain. In the GI tract, I(KACh) could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to beta-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 microM) reversibly activated a Ba(2+)-sensitive and inwardly rectifying K(+) current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I(KACh) current is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers. (+info)
Brief perinatal hypoxia increases severity of pulmonary hypertension after reexposure to hypoxia in infant rats.
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We hypothesized that disrupted alveolarization and lung vascular growth caused by brief perinatal hypoxia would predispose infant rats to higher risk for developing pulmonary hypertension when reexposed to hypoxia. Pregnant rats were exposed to 11% inspired oxygen fraction (barometric pressure, 410 mmHg; inspired oxygen pressure, 76 mmHg) for 3 days before birth and were maintained in hypoxia for 3 days after birth. Control rats were born and raised in room air. At 2 wk of age, rats from both groups were exposed to hypoxia for 1 wk or kept in room air. We found that brief perinatal hypoxia resulted in a greater increase in right ventricular systolic pressure and higher ratio of right ventricle to left ventricle plus septum weights after reexposure to hypoxia after 2 wk of age. Moreover, perinatal hypoxic rats had decreased radial alveolar counts and reduced pulmonary artery density. We conclude that brief perinatal hypoxia increases the severity of pulmonary hypertension when rats are reexposed to hypoxia. We speculate that disrupted alveolarization and lung vascular growth following brief perinatal hypoxia may increase the risk for severe pulmonary hypertension with exposure to adverse stimuli later in life. (+info)
Inactivation properties of human recombinant class E calcium channels.
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The electrophysiological and pharmacological properties of alpha(1E)-containing Ca(2+) channels were investigated by using the patch-clamp technique in the whole cell configuration, in HEK 293 cells stably expressing the human alpha(1E) together with alpha(2b) and beta(1b) accessory subunits. These channels had current-voltage (I-V) characteristics resembling those of high-voltage-activated (HVA) Ca(2+) channels (threshold at -30 mV and peak amplitude at +10 mV in 5 mM Ca(2+)). The currents activated and deactivated with a fast rate, in a time- and voltage-dependent manner. No difference was found in their relative permeability to Ca(2+) and Ba(2+). Inorganic Ca(2+) channel blockers (Cd(2+), Ni(2+)) blocked completely and potently the alpha(1E,)/alpha(2b)delta/beta(1b) mediated currents (IC(50) = 4 and 24.6 microM, respectively). alpha(1E)-mediated currents inactivated rapidly and mainly in a non-Ca(2+)-dependent manner, as evidenced by the fact that 1) decreasing extracellular Ca(2+) from 10 to 2 mM and 2) changing the intracellular concentration of the Ca(2+) chelator 1. 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), did not affect the inactivation characteristics; 3) there was no clear-cut bell-shaped relationship between test potential and inactivation, as would be expected from a Ca(2+)-dependent event. Although Ba(2+) substitution did not affect the inactivation of alpha(1E) channels, Na(+) substitution revealed a small but significant reduction in the extent and rate of inactivation, suggesting that besides the presence of dominant voltage-dependent inactivation, alpha(1E) channels are also affected by a divalent cation-dependent inactivation process. We have analyzed the Ca(2+) currents produced by a range of imposed action potential-like voltage protocols (APVPs). The amplitude and area of the current were dependent on the duration of the waveform employed and were relatively similar to those described for HVA calcium channels. However, the peak latency resembled that obtained for low-voltage-activated (LVA) calcium channels. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca(2+) current amplitude, suggesting an accumulation of inactivation likely to be calcium dependent. The human alpha(1E) gene seems to participate to a Ca(2+) channel type with biophysical and pharmacological properties partly resembling those of LVA and those of HVA channels, with inactivation characteristics more complex than previously believed. (+info)
Low-voltage-activated calcium current does not regulate the firing behavior in paired mechanosensory neurons with different adaptation properties.
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Low-voltage-activated Ca(2+) currents (LVA-I(Ca)) are believed to perform several roles in neurons such as lowering the threshold for action potentials, promoting burst firing and oscillatory behavior, and enhancing synaptic excitation. They also may allow rapid increases in intracellular Ca(2+) concentration. We discovered LVA-I(Ca) in both members of paired mechanoreceptor neurons in a spider, where one neuron adapts rapidly (Type A) and the other slowly (Type B) in response to a step stimulus. To learn if I(Ca) contributed to the difference in adaptation behavior, we studied the kinetics of I(Ca) from isolated somata under single-electrode voltage-clamp and tested its physiological function under current clamp. LVA-I(Ca) was large enough to fire single action potentials when all other voltage-activated currents were blocked, but we found no evidence that it regulated firing behavior. LVA-I(Ca) did not lower the action potential threshold or affect firing frequency. Previous experiments have failed to find Ca(2+)-activated K(+) current (I(K(Ca))) in the somata of these neurons, so it is also unlikely that LVA-I(Ca) interacts with I(K(Ca)) to produce oscillatory behavior. We conclude that LVA-Ca(2+) channels in the somata, and possible in the dendrites, of these neurons open in response to the depolarization caused by receptor current and by the voltage-activated Na(+) current (I(Na)) that produces action potential(s). However, the role of the increased intracellular Ca(2+) concentration in neuronal function remains enigmatic. (+info)
Prototypical imidazoline-1 receptor ligand moxonidine activates alpha2-adrenoceptors in bulbospinal neurons of the RVL.
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Moxonidine is an antihypertensive drug that lowers sympathetic vasomotor tone by stimulating either alpha2-adrenergic (alpha2-AR) or imidazoline I1 receptors within the rostral ventrolateral medulla (RVL). In this study, we investigated the effects of moxonidine (10 microM) on RVL neurons in brain stem slices of neonatal rats. We recorded mainly from retrogradely labeled RVL bulbospinal neurons (putative presympathetic neurons) except for some extracellular recordings. Prazosin was used to block alpha1-adrenoceptors. Moxonidine inhibited the extracellularly recorded discharges of all spontaneously active RVL neurons tested (bulbospinal and unidentified). This effect was reversed or blocked by the selective alpha2-AR antagonist SKF 86466 (10 microM). In contrast, the I1 imidazoline ligand AGN 192403 (10 microM) had no effect on the spontaneous activity. In whole cell recordings (holding potential -70 mV), moxonidine produced a small and variable outward current (mean 7 pA). This current was observed in both tyrosine hydroxylase-immunoreactive and other bulbospinal neurons and was blocked by SKF 86466. Excitatory postsynaptic currents (EPSCs) evoked by focal electrical stimulation were isolated by incubation with gabazine and strychnine, and inhibitory postsynaptic currents (IPSCs) were isolated with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Moxonidine reduced the amplitude of the evoked EPSCs (EC(50) = 1 microM; 53% inhibition at 10 microM) but not their decay time constant (5.6 ms). The effect of moxonidine on EPSCs persisted in barium (300 microM) and was reduced approximately 80% by SKF 86466. Moxonidine also reduced the amplitude of evoked IPSCs by 63%. In conclusion, moxonidine inhibits putative RVL presympathetic neurons both presynaptically and postsynaptically. All observed effects in the present study are consistent with an alpha2-AR agonist activity of moxonidine. (+info)
Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons.
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The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration. (+info)
M-type K+ currents in rat cultured thoracolumbar sympathetic neurones and their role in uracil nucleotide-evoked noradrenaline release.
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Cultured sympathetic neurones are depolarized and release noradrenaline in response to extracellular ATP, UDP and UTP. We examined the possibility that, in neurones cultured from rat thoracolumbar sympathetic ganglia, inhibition of the M-type potassium current might underlie the effects of UDP and UTP. Reverse transcriptase-polymerase chain reaction indicated that the cultured cells contained mRNA for P2Y(2)-, P2Y(4)- and P2Y(6)-receptors as well as for the KCNQ2- and KCNQ3-subunits which have been suggested to assemble into M-channels. In cultures of neurones taken from newborn as well as from 10 day-old rats, oxotremorine, the M-channel blocker Ba(2+) and UDP all released previously stored [(3)H]-noradrenaline. The neurones possessed M-currents, the kinetic properties of which were similar in neurones from newborn and 9 - 12 day-old rats. UDP, UTP and ATP had no effect on M-currents in neurones prepared from newborn rats. Oxotremorine and Ba(2+) substantially inhibited the current. ATP also had no effect on the M-current in neurones prepared from 9 - 12 day-old rats. Oxotremorine and Ba(2+) again caused marked inhibition. In contrast to cultures from newborn animals, UDP and UTP attenuated the M-current in neurones from 9 - 12 day-old rats; however, the maximal inhibition was less than 30%. The results indicate that inhibition of the M-current is not involved in uracil nucleotide-induced transmitter release from rat cultured sympathetic neurones during early development. M-current inhibition may contribute to release at later stages, but only to a minor extent. The mechanism leading to noradrenaline release by UDP and UTP remains unknown. (+info)
Use-dependent facilitation and depression of L-type Ca2+ current in guinea-pig ventricular myocytes: modulation by Ca2+ and isoprenaline.
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OBJECTIVE: An increase in stimulation frequency can facilitate or depress cardiac Ca2+ current (ICa). The aim was to examine the Ca2+ dependence of these effects, to determine if facilitation is sustained, and to elucidate the mechanism by which isoprenaline modulates facilitation. METHODS: We examined the effects of increasing the stimulation frequency for 1 min, from 0.05 to 1 Hz, on ICa recorded from guinea-pig ventricular myocytes, using the whole-cell, voltage-clamp technique. RESULTS: 1 Hz stimulation caused a facilitation of ICa that peaked in 5 s and was followed by depression towards the basal level. Metabolic inhibitors or replacement of extracellular Ca2+ with Ba2+ abolished facilitation without affecting depression, implying that they are independent processes and that facilitation required ATP and Ca2+. Subtraction of the depression observed in either condition, from the response to 1 Hz stimulation recorded under control conditions, revealed that ICa facilitation was well maintained during 1 Hz stimulation. Increased intracellular Ca2+ buffering reduced both phases of the response. Furthermore, varying the extracellular Ca2+ concentration ([Ca2+]o) revealed a Ca(2+)-dependent enhancement of depression and a bell-shaped dependence of facilitation on [Ca2+]o. Facilitation increased with [Ca2+]o up to 1 mM, then declined at higher concentrations due to partial masking by the overlaping depression. Isoprenaline produced concentration-dependent inhibition of facilitation and enhancement of depression when pipettes contained 2 mM EGTA, but not BAPTA. For an equivalent increase in ICa amplitude, the effects of isoprenaline and elevated [Ca2+]o on the response to 1 Hz stimulation were quantitatively the same. CONCLUSIONS: Facilitation is sustained during increased activity, but appears transient due to overlapping depression. Both responses are promoted by increased submembrane [Ca2+]. Isoprenaline appears to modulate facilitation and depression as a consequence of increased Ca2+ influx, rather than cAMP-dependent phosphorylation. The apparent block of facilitation by isoprenaline may result from masking by the enhanced depression. (+info)