Dual pharmacological properties of a cyclic AMP-sensitive potassium channel.
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Bovine adrenal zona fasciculata cells express a novel K+ current (IAC) that sets the resting potential while it couples adrenocorticotropin and angiotensin II receptors to membrane depolarization and cortisol secretion. IAC is distinctive among K+ channels both in its activation by ATP and its inhibition by cyclic AMP. Whole-cell and single-channel patch-clamp recording was used to establish a pharmacological profile of IAC K+ channels. IAC was blocked by antagonists of cyclic nucleotide-gated channels, including the diphenylbutylpiperidine (DPBP) antipsychotic pimozide and l-cis-diltiazem. Other DPBPs, including penfluridol and fluspirilene, also potently inhibited this channel. The inhibition of IAC by DPBPs was selective because 200-fold higher concentrations of penfluridol were required to inhibit voltage-gated IA K+ channels in adrenal zona fasciculata cells. Standard K+ channel antagonists blocked IAC at concentrations 100- to 100,000-fold higher than the DPBPs. IAC channels were also inhibited by the sulfonylureas glyburide and tolbutamide but at concentrations higher than those that typically block ATP-sensitive inward rectifier K+ channels. Overall, the relative order of potency and associated IC50 values for IAC antagonists were as follows: penfluridol (0.187 microM) > fluspirilene (0.232 microM) > pimozide (0.354 microM) >> l-cis-diltiazem (24.9 microM) approximately quinidine (24.1 microM) > bupivacaine (113.2 microM) > tolbutamide (784.4 microM) > BaCl2 (1027 microM) > 4-aminopyridine (2750 microM) > tetraethylammonium (24,270 microM). IAC channels are unique in combining the pharmacological properties of K+-selective channels with those of cyclic nucleotide-gated cation channels. The potent block of IAC channels identifies DPBPs as a new class of K+ channel antagonists and suggests additional targets for these neuroleptics in the central nervous system. (+info)
Probable effect of photoperiod on seasonal variation in the nuclear volume of the adrenal cortex of viscacha (Lagostomus maximus maximus).
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The neuroendocrine system regulates several organic functions such as reproduction, metabolism and adaptation to the environment. This system shows seasonal changes linked to the environment. The experimental model used in the present study was Lagostomus maximus maximus (viscacha). The reproduction of males of this species is photoperiod dependent. Twenty-four adult male viscachas were captured in their habitat at different times during one year. The adrenal glands were processed for light microscopy. Serial cuts were stained with hematoxylin-eosin for the morphometric study, and 100 nuclei of each zone of the adrenal cortex were counted per animal. Data were analyzed statistically by ANOVA and the Tukey test. The cells of the glomerulosa zone are arranged in a tube-shaped structure. The fasciculata zone has large cells with central nuclei and clearly visible nucleoli and with a vacuolar cytoplasm. In the reticularis zone there are two of types of cells, one with a nucleus of fine chromatin and a clearly visible nucleolus and the other with nuclear pycnosis. Morphometric analysis showed maximum nuclear volumes during the February-March period with values of 133 +/- 7.3 microm3 for the glomerulosa, 286.4 +/- 14.72 microm3 for the fasciculata, and 126.3 +/- 9.49 microm3 for the reticularis. Minimum nuclear volumes were observed in August with values of 88.24 +/- 9.9 microm3 for the glomerulosa, 163.7 +/- 7.78 microm3 for the fasciculata and 64.58 +/- 4.53 microm3 for the reticularis. The short winter photoperiod to which viscacha is subjected could inhibit the adrenal cortex through a melatonin increase which reduces the nuclear volume as well as the cellular activity. (+info)
Corticotropin increases protein tyrosine phosphatase activity by a cAMP-dependent mechanism in rat adrenal gland.
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Corticotropin signal transduction pathway involves serine/threonine protein phosphorylation. Recent reports suggest that protein tyrosine dephosphorylation may also be an integral component of that pathway. The present study was performed to investigate the role played by protein tyrosine phosphatases (PTPs) on acute response to corticotropin and the hypothetical regulation of PTPs by this hormone. We have used two powerful cell permeant PTP inhibitors, phenylarsine oxide (PAO) and pervanadate (PV), in order to examine the relevance of PTP activity on hormone-stimulated and 8-bromo-adenosine 3',5'-phosphate (8Br-cAMP is a permeant analogue of adenosine 3',5'-phosphate)-stimulated steroidogenesis in adrenal zona fasciculata (ZF) cells. In both cases, PAO and PV inhibited the steroid production in a dose-dependent fashion, and had no effect on steroidogenesis supported by a permeant analogue of cholesterol. The effect of hormonal stimulation on PTP activity was analyzed in rat adrenal ZF. In vivo corticotropin treatment reduced phosphotyrosine content in endogenous proteins and produced a transient increase of PTP activity in the cytosolic fraction, reaching a maximum (twofold) after 15 min. Incubation of adrenal ZF with 8Br-cAMP also produced PTP activation, suggesting that it can be mediated by cAMP-dependent protein kinase (PKA)-dependent phosphorylation. Detection of PTP activity in an in-gel assay showed three corticotropin-stimulated soluble PTPs with molecular masses of 115, 80 and 50 kDa. In summary, we report for the first time a hormone-dependent PTP activation in a steroidogenic tissue and provide evidence that PTP activity plays an important role in corticotropin signal pathway, acting downstream of PKA activation and upstream of cholesterol transport across the mitochondrial membrane. (+info)
Characterisation of the muscarinic receptor subtype M3 in the bovine zona fasciculata-reticularis cells by receptor binding, mRNA and functional studies.
(4/90)
In the present work we have investigated which muscarinic (M) receptor subtype is responsible for the steroidogenic effect of muscarinic agonists in bovine zona fasciculata-reticularis (ZFR) cells in culture. Radioligand binding studies using the muscarinic antagonist [(3)H]quinuclidinyl benzilate ([(3)H]QNB) demonstrated binding sites of high affinity (K(d)=0.45 nM) and low capacity ( approximately 8000 sites/cell). Pharmacological characterisation of muscarinic receptors was assessed by evaluating the effects of the M(3)>M(1)>>M(2) antagonist 4-DAMP (4-diphenylacetyl-N-methylpiperidine) and the M(1)=M(4)> M(3)>>M(2) antagonist pirenzepine on the binding of [(3)H]QNB and carbachol-induced cortisol production. For both parameters, the potency of 4-DAMP was about two orders of magnitude higher than that of pirenzepine. Reverse transcriptase (RT)-PCR analysis of bovine ZFR mRNAs using specific primers for M(2), M(3) and M(4) receptors revealed the expression of only M(3) mRNA. Moreover, carbachol significantly stimulated inositol phosphate accumulation, but had no inhibitory effect on basal or ACTH-induced cAMP production. Indeed, carbachol potentiated ACTH-induced cAMP production and this effect was, in part, mediated through protein kinase C. Lastly, neomycin, an inhibitor of phosphoinositide turnover, significantly attenuated carbachol-evoked cortisol production. Thus, pharmacological, biochemical and mRNA studies indicate that the M(3) receptor subtype is responsible for the biological effects of muscarinic agonists in bovine ZFR cells. (+info)
Adenosine inhibits a non-inactivating K+ current in bovine adrenal cortical cells by activation of multiple P1 receptors.
(5/90)
1. Bovine adrenal zona fasciculata (AZF) cells express a non-inactivating K+ current (IAC) that sets the resting potential while it is activated by intracellular ATP. In whole-cell patch clamp recordings from bovine AZF cells, we found that adenosine selectively inhibited IAC by a maximum of 78.4 +/- 4.6 % (n = 8) with an IC50 of 71 nM. The non-selective adenosine receptor agonist NECA effectively inhibited IAC by 79.3 +/- 2.9 % (n = 24) at a concentration of 100 nM. 2. Inhibition of IAC was mediated through multiple P1 adenosine receptor subtypes. The A1-selective agonist CCPA (10 nM), the A2A-selective agonist CGS 21680 (100 nM) and the A3-selective agonist IB-MECA (10 nM) inhibited IAC by 64.8 +/- 8.4, 78.4 +/- 4.6 and 69.3 +/- 6.9 %, respectively. 3. Specific adenosine receptor subtype antagonists including DPCPX (A1), ZM 241385 (A2A) and MRS 1191 (A3) effectively blocked inhibition of IAC by adenosine receptor-selective agonists. 4. A mixture of the three adenosine receptor antagonists completely suppressed inhibition of IAC by adenosine, but failed to alter inhibition by external ATP which acts through a separate P2 nucleotide receptor. 5. Inhibition of IAC by adenosine or NECA was eliminated by substituting GDP-beta-S for GTP in the pipette, or by replacing ATP with AMP-PNP or UTP. 6. In addition to inhibiting IAC, adenosine (10 microM) depolarized AZF cells by 46.2 +/- 5.8 mV (n = 6). 7. These results show that bovine AZF cells express at least three adenosine receptor subtypes (A1, A2A, A3), each of which is coupled to the inhibition of IAC K+ channels through a G-protein-dependent mechanism requiring ATP hydrolysis. Adenosine-mediated inhibition of IAC is associated with membrane depolarization. Adenosine and other purines may co-ordinate the stress-induced secretion of corticosteroids and catecholamines from the adrenal gland. (+info)
Mibefradil potently blocks ATP-activated K(+) channels in adrenal cells.
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Mibefradil is a novel Ca(2+) channel antagonist that preferentially blocks T-type Ca(2+) channels in many cells. Using whole-cell and single-channel patch-clamp recording, we found that mibefradil also potently blocked an ATP-activated K(+) channel (I(AC)) expressed by adrenal zona fasciculata cells. I(AC) channels were inhibited by mibefradil with an IC(50) value of 0.50 microM, a concentration 2-fold lower than that required to inhibit T-type Ca(2+) channels under similar conditions in the same cells. The inhibition of I(AC) by mibefradil was independent of the membrane potential. Mibefradil also reversibly blocked, with similar potency, unitary I(AC) currents recorded in outside-out membrane patches. An analysis of dwell time histograms indicated the presence of two closed and one open state. Mibefradil (1 microM) increased the duration of the two closed time constants (tau(c1) and tau(c2)) from 2.30 +/- 0.18 and 27.9 +/- 4.7 ms to 4.32 +/- 0.61 and 62.5 +/- 13.8 ms, respectively, but did not alter the open time constant (tau(o)). Mibefradil also failed to reduce the size of the unitary I(AC) current. A voltage-gated A-type K(+) current was also inhibited by mibefradil at concentrations approximately 10-fold higher than those required to block I(AC) (IC(50) = 4.65 microM). These results identify mibefradil as a potent inhibitor of ATP-activated K(+) channels in adrenal zona fasciculata cells. It appears to function by stabilizing closed states of these channels. In contrast to its selective block of T-type Ca(2+) channels, mibefradil may be a potent but less-selective K(+) channel blocker. In this regard, the block of K(+) channels may produce some of the toxicity associated with mibefradil in cardiovascular pharmacology. (+info)
Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca(2+) channels in cortisol-secreting cells.
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We have studied the effect of the Ca(2+) antagonist mibefradil on low voltage-activated T-type Ca(2+) channels in whole-cell patch clamp recordings from bovine adrenal zona fasciculata (AZF) cells. AZF cells are distinctive in expressing only T-type Ca(2+) channels, allowing the mechanism of pharmacological agents to be explored without interference from other Ca(2+) channels. The inhibition of T-type Ca(2+) channels by mibefradil was voltage- and use-dependent. When Ca(2+) currents were activated from holding potentials of -80 and -60 mV, mibefradil inhibited currents with IC(50) values of 1.0 and 0.17 microM, respectively. When T-type Ca(2+) current (I(T)) was activated from a holding potential of -90 mV in the presence of 2 microM mibefradil, a single voltage step to -10 mV inhibited I(T) by 16.2% +/- 2.9% (n = 10). With subsequent voltage steps, applied at 10-s intervals, block reached a steady-state value of 51.9% +/- 5.0% (n = 5). Mibefradil (1 microM) produced a leftward shift of 5.7 mV (n = 4) in the voltage-dependent steady-state availability curve such that T-type Ca(2+) channels inactivated at more negative potentials, but this drug did not change the voltage-dependence of T channel opening. Mibefradil failed to alter the kinetics of T channel activation, inactivation, or deactivation, but markedly slowed T channel recovery following an inactivating prepulse. Mibefradil inhibited adrenocorticotropin-stimulated cortisol secretion from AZF cells with an IC(50) value of 3.5 microM. These results show that mibefradil is a relatively potent antagonist of T-type Ca(2+) channels in cortisol-secreting cells. The enhanced potency of mibefradil with sustained or repetitive depolarizations, its shifting of the steady-state inactivation curve, and its slowing of recovery all indicate that this drug preferentially interacts with Ca(2+) channels in the open or inactivated state. The inhibition of cortisol secretion by mibefradil at concentrations similar to those that block I(T) is consistent with a requirement for these channels in corticosteroidogenesis. (+info)
Lack of carcinogenicity of chlorpyrifos insecticide in a high-dose, 2-year dietary toxicity study in Fischer 344 rats.
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Chlorpyrifos (CPF) was administered daily in the feed to evaluate toxicity and oncogenicity potential in male and female Fischer 344 rats, according to U.S. EPA guidelines. Doses for the 2-year study were based on findings in a 13-week feeding study in which lower body weights, urinary perineal staining, adrenal cortical vacuolization, and inhibition (slightly more than 60%) of brain cholinesterase (ChE) occurred at 15 mg/kg/day. The high dose in the subsequent 2-year study was 10 mg/kg/day, with lower doses of 0, 0.05, 0.1, or 1.0 mg/kg/day chosen to define dose-response patterns. Rats given 10 mg/kg/day for 2 years were healthy and there was no evidence of premature deaths. Mild toxicity occurred only in rats given 10 mg/kg/day and consisted of perineal urine soiling in females and a 6-8% body-weight decrease in males. Males given 10 mg/kg/day also had increased adrenal weights and vacuolation of the adrenal zona fasciculata. ChE was considered a measure of exposure. Plasma, RBC, and brain ChE activities were inhibited in rats given 10 mg/kg/day, and the plasma and RBC ChE activities were inhibited in rats given 1.0 mg/kg/day. Chronic exposure to 0.1 mg/kg/day was considered a threshold exposure level for inhibition of plasma ChE. Rats given 10 mg/kg/day, considered a maximum-tolerated dose, had approximately 60% chronic inhibition of brain ChE. This group had similar numbers and types of neoplasms as control rats. Consequently, CPF was not carcinogenic at dose levels up to 10 mg/kg/day. (+info)