Calcium oscillations in freshly isolated neonatal rat cortical neurons.
(33/220)
AIM: To investigate the mechanisms of intracellular calcium concentration ([Ca2+]i) oscillations in freshly isolated neonatal rat cortical neurons. METHODS: Cortical neurons were isolated from neonatal rats 6-7 d after birth by enzymatic digestion. [Ca2+]i changes were measured in a microscopic calcium measurement system with Fura-2 as indicator. RESULTS: In a total of 82 neurons recorded, 47 showed spontaneous [Ca2+]i oscillations. The spontaneous [Ca2+]i oscillations were dependent on [Ca2+]o. Removal of [Ca2+]o completely abolished spontaneous oscillations. Tetraethylammonium 1 mmol/L increased both the amplitude and frequency of calcium oscillations, whereas the frequency was increased by Cs+ 1 mmol/L. Ba2+ 1 mmol/L, in contrast, induced [Ca2+]i oscillations superimposed on a sustained phasic increase. CONCLUSION: Spontaneous [Ca2+]i oscillation is an intrinsic property of neonatal rat cortical neurons. Potassium channels play an important role in the control of both the amplitude and frequency of [Ca2+]i oscillations in cortical neurons. (+info)
Interaction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserve.
(34/220)
1 The aim of this study was to investigate the possible role of the interaction of different potassium channels in dog ventricular muscle, by applying the conventional microelectrode and whole cell patch-clamp techniques at 37 degrees C. 2 Complete block of I(Kr) by 1 micro M dofetilide lengthened action potential duration (APD) by 45.6+/-3.6% at 0.2 Hz (n=13). Chromanol 293B applied alone at 10 micro M (a concentration which selectively blocks I(Ks)) did not markedly lengthen APD (<7%), but when repolarization had already been prolonged by complete I(Kr) block with 1 micro M dofetilide, inhibition of I(Ks) with 10 micro M chromanol 293B substantially delayed repolarization by 38.5+/-8.2% at 0.2 Hz (n=6). 3 BaCl(2), at a concentration of 10 micro M which blocks I(Kl) without affecting other currents, lengthened APD by 33.0+/-3.1% (n=11), but when I(Kr) was blocked with 1 micro M dofetilide, 10 micro M BaCl(2) produced a more excessive rate dependent lengthening in APD, frequently (in three out of seven preparations) initiating early afterdepolarizations. 4 These findings indicate that if only one type of potassium channels is inhibited in dog ventricular muscle, excessive APD lengthening is not likely to occur. Dog ventricular myocytes seem to repolarize with a strong safety margin ('repolarization reserve'). However, when this normal 'repolarization reserve' is attenuated, otherwise minimal or moderate potassium current inhibition can result in excessive and potentially proarrhythmic prolongation of the ventricular APD. Therefore, application of drugs which are able to block more than one type of potassium channel is probably more hazardous than the use of a specific inhibitor of one given sort of potassium channel, and when simultaneous blockade of several kinds of potassium channel may be presumed, a detailed study is needed to define the determinants of 'repolarization reserve'. (+info)
Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning.
(35/220)
A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity. (+info)
Characterization of regulatory volume decrease in freshly isolated mouse cholangiocytes.
(36/220)
Cell volume regulation plays a vital role in many cell functions. Recent study indicates that both K(+) and Cl(-) channels are important for the regulatory volume decrease (RVD) of cholangiocarcinoma cells, but its physiological significance is unclear due to the tumorous nature of the cells used. This present study reports the RVD of normal mouse cholangiocytes by using freshly isolated bile duct cell clusters (BDCC). A relatively simple and practical method of measuring the cross-sectional area of BDCCs by quantitative videomicroscopy was used to indirectly measure their volumes. Mouse cholangiocytes exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate, DIDS, and glibenclamide, suggesting its dependence on certain chloride channels, such as volume-activated chloride channels. It is also inhibited by barium chloride but not by tetraethylammonium chloride, indicating its dependence on certain potassium channels. However, cAMP agonists had no significant effect on the RVD of BDCCs. This indirect method described can be used to study the RVD of cholangiocytes from normal as well as genetically altered mouse livers. (+info)
Growth hormone-releasing peptide-2 reduces inward rectifying K+ currents via a PKA-cAMP-mediated signalling pathway in ovine somatotropes.
(37/220)
Inward-rectifying potassium (Kir) channels are essential for maintaining the resting membrane potential near the K(+) equilibrium and they are responsible for hyperpolarisation-induced K(+) influx. We characterised the Kir current in primary cultured ovine somatotropes and examined the effect of growth hormone-releasing peptide-2 (GHRP-2) on this current and its related intracellular signalling pathways. The Kir current was, in most cases, isolated using nystatin-perforated patch-clamp techniques. In bath solution containing 5 mM K(+), the Kir current was composed of both transient (fast activated) and delayed (slowly activated) components. An increase in the external K(+) concentration from 5 to 25 mM induced an augmentation of approximately 4-fold in the delayed part of the Kir current and both BaCl(2) and CsCl dose-dependently inhibited this current, confirming the presence of the Kir current in ovine somatotropes. Moreover, this specific effect of high K(+) on the Kir current was only observed in the cells that showed positive staining with anti-growth hormone (GH) antibodies, or in GC cells that belong to a rat somatotrope cell line. Application of GHRP-2 (100 nM) reversibly and significantly reduced the Kir current in bath solutions with 5 or 25 mM K(+) in ovine somatotropes. In addition, we found that the reduction in the Kir current mediated by GHRP-2 was totally abolished by the pretreatments with H89 (1 microM) or Rp-cAMP (100 microM) or by intracellular dialysis of a specific protein kinase A (PKA) inhibitory peptide PKI (10 microM). The specific PKC blocker chelerythrine (1 microM) or inhibitory peptide PKC(19-36) (10 microM) did not show any effects on the GHRP-2-induced decrease in the Kir current. These results suggest that the inhibition of Kir current through PKA-cAMP pathways may play an integral role in GHRP-2-induced depolarisation and GH release in ovine somatotropes. (+info)
Norepinephrine evoked by potassium depolarization increases interstitial adenosine concentration via activation of ecto-5'-nucleotidase in rat hearts.
(38/220)
We examined whether the increase of the extracellular potassium ion concentration, [K(+)](o), can increase the production of interstitial adenosine in the ventricular myocardium, with the use of microdialysis techniques in in situ rat hearts. A microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts, and the tissue in the vicinity of the dialysis was perfused with Tyrode's solution containing AMP through the dialysis probe at a rate of 1.0 microl/min to assess the activity of ecto-5'-nucleotidase. When the K(+) concentration of the perfusate ([K(+)](o)) was increased stepwise from 5.4 mM (control) to up to 140.4 mM, the level of dialysate adenosine significantly increased, in a [K(+)](o)-dependent manner. The presence of CsCl or BaCl(2) (20 mM), which markedly depolarized the resting potential, significantly increased the level of adenosine in the dialysate. Equivalent increases in the osmotic concentration of the perfusate, made by adding sucrose (270 mM), did not change the dialysate adenosine concentration. Introduction of high [K(+)](o) (140.4 mM) significantly increased the level of norepinephrine (NE) in the dialysate, and this increase was abolished in the reserpinized rats hearts. In the presence of an antagonist of alpha(1)-adrenoceptor (prazosin, 50 microM) or protein kinase C (PKC) (chelerythrine, 10 microM) and in reserpinized rats, an introduction of high [K(+)](o) failed to increase the AMP-primed dialysate adenosine concentration. We conclude that high [K(+)](o)-induced NE release from sympathetic nerve terminals increases adenosine by stimulating the PKC-ecto-5'-nucleotidase cascade through alpha(1)-adrenoceptors. (+info)
Development of inward rectification and control of membrane excitability in mesencephalic v neurons.
(39/220)
The present study was performed to assess the postnatal development and functional roles of inward rectifying currents in rat mesencephalic trigeminal (Mes V) neurons, which are involved in the genesis and control of oral-motor activities. Whole cell voltage-clamp recordings obtained from Mes V neurons in brain stem slices identified fast (I(KIR)) and slow (I(h)) inward rectifying currents, which were specifically blocked by BaCl(2) (300-500 microM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288, 10 microM), respectively. The whole cell current density for these channels increased between postnatal days 2 to 12 (P2-P12), and the time courses for I(h) activation and deactivation were each well described by two time constants. Application of ZD 7288 produced membrane hyperpolarization in the majority of cells and prolonged afterhyperpolarization repolarization. Additionally, in the presence of ZD 7288, spike frequency was decreased and adaptation was more pronounced. Interestingly, these neurons exhibited a voltage-dependent membrane resonance (<10 Hz) that was prominent around resting potential and more negative to rest and was blocked by ZD 7288. These results suggest that I(h) contributes to stabilizing resting membrane potential and controlling cell excitability. The presence of I(h) imparts the neuron with the unique property of low-frequency membrane resonance; the ability to discriminate between synaptic inputs based on frequency content. (+info)
Kappa-opioid receptor-mediated enhancement of the hyperpolarization-activated current (I(h)) through mobilization of intracellular calcium in rat nucleus raphe magnus.
(40/220)
The hyperpolarization-activated current (Ih) is important in the control of resting membrane potential, in the regulation of network firing pattern and in the modulation of presynaptic transmitter release in central neurons. Recent studies on native and cloned Ih channels have demonstrated that the Ih channel is commonly modulated by cAMP through a positive shift in its voltage dependence without a change in its maximum current. The present study demonstrates that activation of kappa-opioid receptors enhances Ih by increasing its maximum current in brainstem neurons in the nucleus raphe magnus. Agents that interfere with the release of intracellular calcium from calcium stores altered the maximum Ih and significantly attenuated the kappa-receptor-mediated enhancement of Ih. These results suggest that kappa-opioid receptors enhance the maximum Ih by mobilizing intracellular calcium from calcium stores. This provides a physiological function for kappa-receptor-stimulated calcium release and may suggest another Ih-regulating mechanism by intracellular calcium in central neurons. (+info)