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R A Wilke

Publications and source records attributed to R A Wilke.

18 recordsLinked to original sources
PubMed2000

Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release.

Adenosine Triphosphate↗
PubMed2000

Novel human TEF-1 isoforms exhibit altered DNA binding and functional properties.

The transcriptional enhancer factor-1 (TEF-1) is a member of the TEA/ATTS domain family. TEF-1 binds to GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and M-CAT (GGTATG) response elements and is involved in the transactivation of a variety of genes, including the SV40 large T antigen, mammalian muscle-specific genes, and human chorionic somatomammotropin genes. Also, TEF-1 acts as a transcriptional repressor in placental cells, possibly through interaction with the TATA binding protein (TBP), preventing TBP binding to the TATA box. Here we describe the cloning, tissue-specific expression pattern, and functional characterization of two novel TEF-1 isoforms, TEF-1beta and TEF-1gamma. These isoforms most likely arise from alternative splicing of mRNA transcribed from a single gene and involve substitutions and/or insertions in a region immediately following the DNA binding domain. TEF-1beta appears to be widely distributed like the prototypic TEF-1, designated TEF-1alpha, whereas TEF-1gamma exhibits a narrower tissue-specific expression pattern that includes pancreas, kidney, and skeletal and heart muscle. The relatively limited sequence alterations among these isoforms cause significant changes in their DNA binding and transcriptional activities. TEF-1beta and TEF-1gamma bind to GT-IIC sequences with higher affinity and repress hCS promoter more efficiently than TEF-1alpha. These results suggest that each TEF-1 isoform may play unique regulatory roles in various tissues.

Alternative Splicing↗
PubMed2000

Are pesticides really endocrine disruptors?

In recent years, a considerable amount of basic laboratory investigation has been targeted toward understanding the relationship between environmental contaminants and cellular endocrine function. A number of environmental contaminants are now known to alter endocrine physiology without acting as classic toxicants. Hence, a new field of inquiry has emerged within the discipline of environmental toxicology, the study of endocrine disrupting chemicals (EDCs). Pesticides represent one of the better studied groups of EDCs. As the tools of molecular biology become increasingly sophisticated, our ability to understand the endocrine effects of these compounds continues to broaden at a remarkable rate. Still, clinical data linking them to derangements in human endocrine function have been quite limited. While the scientific community awaits further epidemiological assessment of the impact of pesticides on public health, responsible land management practices should be employed in an effort to reduce the burden of these chemicals ultimately reaching our water supply.

Endocrine System↗
PubMed1999

Sigma receptor photolabeling and sigma receptor-mediated modulation of potassium channels in tumor cells.

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (IK). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in IK, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 microM. SKF10047 also inhibited IK with a similar EC50 (14 microM). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5'-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.

Antipsychotic Agents↗
PubMed1999

K+ channel modulation in rodent neurohypophysial nerve terminals by sigma receptors and not by dopamine receptors.

1. Sigma receptors bind a diverse group of chemically unrelated ligands, including pentazocine, apomorphine (a dopamine receptor agonist) and haloperidol (a dopamine receptor antagonist). Although sigma binding sites are widely distributed, their physiological roles are poorly understood. Here, the whole-terminal patch-clamp technique was used to demonstrate that sigma receptors modulate K+ channels in rodent neurohypophysis. 2. Previous work suggested that dopamine type 4 (D4) receptors modulate neurohypophysial K+ current, so this study initially tested the role of dopamine receptors. Experiments using transgenic mice lacking D2, D3 or D4 receptors indicated that the reduction of K+ current by PPHT and U101958 (ligands thought to be selective for dopamine receptors) is not mediated by dopamine receptors. The sensitivity of the response to U101958 (a drug that binds to D4 receptors) was the same in both wild-type and D4 receptor-deficient mice. 3. Experiments with other ligands revealed a pharmacological signature inconsistent with any known dopamine receptor. Furthermore, dopamine itself (at 100 microM) had no effect. Thus, despite the activity of a number of putative dopamine receptor ligands, dopamine receptors play no role in the modulation of neurohypophysial K+ channels. 4. Because of the negative results regarding dopamine receptors, and because some of the dopamine receptors ligands used here are known to bind also to sigma receptors, experiments were conducted to test for the involvement of sigma receptors. In rat neurohypophysis the sigma receptor ligands SKF10047, pentazocine, and ditolylguanidine all reversibly inhibited K+ current in a concentration-dependent fashion, as did haloperidol and apomorphine (ligands that bind to both dopamine and sigma receptors). The activity of these and other ligands tested here matches the reported binding specificity for sigma receptors. 5. Fifteen candidate endogenous sigma receptor ligands, including biogenic amines (e.g dopamine and serotonin), steroids (e.g. progesterone), and peptides (e.g. neuropeptide Y), were screened for activity at the sigma receptor. All were without effect. 6. Haloperidol reduced K+ current proportionally at all voltages without shifting the voltage dependence of activation and inactivation. Sigma receptor ligands inhibited current through two distinct K+ channels, the A-channel and the Ca2+-dependent K+ channel. In rat, all drugs reduced current through both channels proportionally, suggesting that both channels are modulated by a single population of sigma receptors. In contrast, mouse peptidergic nerve terminals either have two receptors which are sensitive to these drugs, or a single receptor that is differentially coupled to ion channel function. 7. The inhibition of voltage-activated K+ current by sigma receptors would be expected to enhance the secretion of oxytocin and vasopressin from the neurohypophysis.

Animals↗
PubMed1998

Membrane excitability in the neurohypophysis.

The application of patch clamp technology to the neurohypophysis has contributed significantly to our understanding of the membrane events governing neuropeptide secretion. Nerve terminals within the posterior pituitary are now known to contain three distinct K+ channel subtypes, a rapidly inactivated channel (responsible for A current), a Ca(2+)-activated K+ channel, and a delayed rectifier channel. Activation of a D2 subtype dopamine receptor reduces both the A-current and current through the Ca2+ activated K+ channels. These actions can be expected to enhance neuropeptide release. Release of nitric oxide reduces the amplitude of the A-current but enhances current through the Ca(2+)-activated K+ channel, and this would provide mechanisms for more complex modes of regulation of release. Neurohypophysial nerve terminals also express at least two types of Ca2+ channels. The first is a dihydropyridine-sensitive, "L-type" channel. The second resembles the "N-type" Ca2+ channel. Patch clamp recordings have shown that tissue culture medium conditioned by exposure to T-cells enhances this Ca2+ current. This may represent a mechanistic link between activation of the immune system and functional membrane changes within the neurohypophysis. GABA-activated Cl- channels have also been described within the neurohypophysis, and these receptors can be modulated by neuroactive steroids. One of these, the progesterone derivative allopregnanolone, changes dramatically during female reproductive transitions. Such an interaction could represent a pivotal mechanistic step in the onset of parturition, and the neurohypophysial GABA receptor may hold promise as a target of therapeutic intervention in clinical cases of preterm labor.

Action Potentials↗
PubMed1998

Dopamine D4 receptor mediated inhibition of potassium current in neurohypophysial nerve terminals.

Dopamine influences the release of neurohypophysial peptides in vivo. However, the extent to which this effect is caused by a direct dopaminergic action within the neurohypophysis remains unclear. With use of the patch-clamp technique on thin slices of rat posterior pituitary glands, we now provide evidence that dopaminergic agonists inhibit potassium current (IK) in neurohypophysial nerve terminals. Superfusion with the dopamine receptor agonist, (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin (PPHT), causes a reversible inhibition of whole-terminal IK under voltage clamp. This effect is concentration-dependent, with a maximal inhibition of 40 +/- 5% and an EC50 of 1.8 +/- 1.0 microM. It can be blocked with either a nonselective D2-like antagonist (100 microM eticlopride) or with the highly selective D4 antagonist, RBI-257 (10 microM). U101958 (a derivative of RBI-257) exhibits agonist activity similar to PPHT. Neither SKF 38393 (a D1/D5 agonist) nor quinpirole (a D2/D3 agonist) had any effect on whole-terminal IK in this preparation. Kinetic analysis demonstrated that the amplitude of both the rapidly and slowly inactivating phases of neurohypophysial IK are reduced by D4 receptor activation. These two separate current components have previously been shown to represent current through two distinct potassium channels, an A-current channel and a high-conductance Ca++-activated K+ channel. Thus, both channel types can be modulated by D4 receptors. This effect is likely to enhance the release of neurohypophysial peptides in vivo.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1994

Decreased adrenal medullary catecholamine release in spontaneously diabetic BB-Wistar rats. Role of hypoglycemia.

We have demonstrated previously that spontaneously diabetic BB-Wistar rats exhibit decreased adrenal medullary catecholamine secretion in response to splanchnic nerve terminal stimulation. We hypothesized that this abnormality is caused by changes in the sensitivity of the adrenomedullary chromaffin cells to acetylcholine (ACh). To study this hypothesis, we isolated adrenal glands from control and spontaneously diabetic BB-Wistar rats, perfused them with ACh, and measured catecholamine secretion. Adrenal catecholamine release in response to ACh was significantly decreased at 2, 8, and 16 weeks after the onset of diabetes compared with age-matched, nondiabetic control rats. Catecholamine release in response to perfusion with 20 mM K+ was the same in adrenals from diabetic and control rats. The decreased responsiveness of diabetic rat adrenals to perfusion with ACh was significantly correlated with a decrease in the release of catecholamines in response to splanchnic nerve stimulation. A similar defect in catecholamine secretion was also seen in adrenals harvested from nondiabetic BB-Wistar rats following a 3-h period of acute hypoglycemia; however, the adrenal response to potassium was also decreased as was the catecholamine content of the adrenal. Conversely, nondiabetic BB-Wistar rats made diabetic with streptozocin (STZ) and maintained in a hyperglycemic state did not exhibit catecholamine hyposecretion 2 weeks after STZ administration. Collectively, the data describe decreased adrenomedullary response to cholinergic stimulation in spontaneously diabetic rats as early as 2 weeks after the onset of diabetes and that a similar, although more severe, hyposecretion occurs after acute, severe hypoglycemia.

Acetylcholine↗
PubMed1993

Decreased catecholamine secretion from the adrenal medullae of chronically diabetic BB-Wistar rats.

Many humans with IDDM eventually lose the capacity to secrete epinephrine from their adrenal medullae. The mechanism for this pathological change is unknown. We hypothesized that this abnormality is attributable to neuropathic changes in the greater splanchnic nerves or in the chromaffin cells that they innervate. To study this hypothesis, we isolated rat adrenal glands, perfused them ex vivo, and measured the epinephrine content of the perfusate under various conditions of stimulation. We used transmural electrical stimulation (20-80 V, at 10 Hz) to induce epinephrine secretion indirectly by selectively activating residual splanchnic nerve terminals within the isolated glands. Under these conditions, epinephrine secretion was severely attenuated in glands from female BB-Wistar rats with diabetes of 4 mo duration compared with their age-matched, nondiabetic controls. These perfused diabetic adrenal medullae also demonstrated decreased catecholamine release in response to direct chromaffin cell depolarization with 20 mM K+, evidence that a functional alteration exists within the chromaffin cells themselves. Nonetheless, total catecholamine content of adrenal medullae from these diabetic rats was not significantly different from controls, indicating that the secretory defect was not simply attributable to a difference in the amount of catecholamines stored and available for release. Herein, we also provide histological evidence of degenerative changes within the cholinergic nerve terminals that innervate these glands.

Adrenal Medulla↗
PubMed1992

Histochemical discrimination of fibers in regenerating rat infraorbital nerve.

In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.

Animals↗
PubMed1989

Effect of KT-362, a putative intracellular calcium antagonist, on norepinephrine-induced contractions and inositol monophosphate accumulation in canine femoral artery.

The purpose of the present study was to determine if the intracellular calcium antagonist, KT-362, inhibits norepinephrine- (NE) induced contractions and inositol monophosphate (IP) accumulation in canine femoral arteries preincubated with [3H]inositol. Norepinephrine dose-dependently increased contractile tension and produced a parallel stimulation of inositol phospholipid hydrolysis as measured by IP accumulation. This stimulation was inhibited by the selective alpha 1 adrenoceptor antagonist, prazosin (0.1 microM), indicating that the NE-induced stimulation of inositol phospholipid hydrolysis is coupled to alpha 1 adrenoceptor activation in canine femoral artery. Pretreatment with nitroglycerin (100 microM), 8-Br cyclic guanosine monophosphate (cGMP) (1 microM), or diltiazem (40 microM) inhibited contractile responses produced by NE; however, these agents had no significant effect on NE-induced IP accumulation. In contrast, pretreatment with KT-362 (10-100 microM) greatly inhibited both the NE-induced contractions and IP accumulation. KT-362 also produced a marked inhibition of NE-induced contractions in normal as well as zero calcium buffer, whereas diltiazem (40 microM) had no effect in zero calcium buffer. These results indicate that the mechanism of action of KT-362 differs from diltiazem, nitroglycerin, and 8-Br cGMP, and these data suggest that one of the mechanisms by which KT-362 antagonizes NE-induced vasoconstrictor responses is by decreasing inositol phospholipid hydrolysis in canine femoral artery.

Animals↗