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Biomedical subjects

F T Crews

Publications and source records attributed to F T Crews.

At least 73 records · Page 4Linked to original sources
PubMed1990

Adenosine and its analogs stimulate phosphoinositide hydrolysis in the kidney.

Renal blood flow, glomerular filtration rate and sodium excretion are known to be affected by adenosine. The present studies were undertaken to investigate the actions of adenosine and its analogs (both agonists and antagonists) on phosphoinositide (PI) hydrolysis in the outer medullary slices. Adenosine was found to cause a dose-dependent stimulation of PI hydrolysis (ED50, 2.8 microM) in renal slices from outer medulla. The adenosine analogs 5'-(N-cyclopropyl)-carboxamidoadenosine (NCCA) and 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated PI hydrolysis in renal medulla. Stimulation of PI hydrolysis was blocked by the adenosine antagonists: aminophylline, 1,3-dipropyl-7-methylxanthine (DMX) and 8-(p-sulfophenyl)-theophylline (8-SPT). Caffeine not only antagonized adenosine-stimulated PI hydrolysis but also increased PI hydrolysis independently. These results indicate that adenosine stimulates PI hydrolysis in renal medulla through a receptor-mediated mechanism.

Adenosine↗
PubMed1990

Desensitization of muscarinic stimulated hippocampal cell firing is related to phosphoinositide hydrolysis and inhibited by lithium.

The potency and efficacy of a series of muscarinic agonists for stimulation of neuronal firing rate was compared with stimulation of phosphoinositide (PPI) hydrolysis in similar hippocampal slice preparations. Carbachol, muscarine, pilocarpine, arecoline, bethanechol and oxotremorine varied in potency, but stimulated neuronal firing to a similar extent. At higher concentrations, all of the drugs except oxotremorine caused a decrease in firing rate (desensitization). A comparison of the concentration-response curves for PPI hydrolysis and neuronal firing rates showed that desensitization occurred at a threshold level of PPI hydrolysis. Although the concentration of drug that caused the decrease in firing rate was different for each agonist, the level of PPI hydrolysis at the desensitizing concentration was similar, with the exception of oxotremorine. Oxotremorine, the weakest agonist for stimulation of PPI hydrolysis, did not reach this PPI hydrolysis threshold and did not exhibit desensitization. Oxotremorine was also capable of both blocking and reversing the desensitization caused by carbachol. Low concentrations of pirenzepine, an M1 selective muscarinic antagonist, reversed carbachol desensitization. Concentrations of lithium that disrupt the phosphoinositide cycle by preventing recycling of free inositol (Allison, J. H., et al.: Biochem. Biophys. Res. Commun. 71: 664-670, 1976; Hallcher, L. M. and Sherman, W. R.: J. Biol. Chem. 225: 10896-10901, 1980) slowly reversed desensitization. Furthermore, inositol added to the buffer could re-establish desensitization in lithium-treated preparations. These studies suggest that muscarinic desensitization of hippocampal cell firing is related to large increases in phosphoinositide hydrolysis. Muscarinic receptor-stimulated increases in cell firing may be mediated by a subtype or state of muscarinic receptor different from that mediating phosphoinositide hydrolysis and desensitization.

Action Potentials↗
PubMed1989

Cholinergic receptors in renal medullary collecting duct cells.

Intrarenal administration of cholinergic agents produces diuresis. However, neither cholinergic innervation or specific cholinergic receptors have been shown to be present in the kidney. Recently, we have demonstrated that carbachol, a cholinergic agent, stimulates phosphoinositide hydrolysis in the inner medullary collecting duct (IMCD) cells. The effect was blocked by atropine (a cholinergic antagonist), suggesting that phosphoinositide hydrolysis occurs through the interaction of carbachol with specific cholinergic receptors in these cells. Therefore, we examined the cholinergic receptors in IMCD cells by measurement of radioligand binding of a cholinergic receptor antagonist, I-quinuclidinyl (phenyl-4-3H)benzilate([3H]QNB). The IMCD cells were prepared from rabbit kidneys by incubating the inner medullary slices with collagenase and treating the isolated cells with hypotonic solution to lyse cells other than IMCD cells. Binding of [3H]QNB to IMCD cells was measured at 37 degrees C for 60 min in the absence (total binding) and the presence (nonspecific binding) of 100 microM atropine (a muscarinic receptor antagonist). The specific binding (the difference between total and nonspecific binding) of [3H]QNB to IMCD cells was saturable with a Bmax (maximum binding sites) of 27.5 fmol/mg of protein and Kd (dissociation constant) of 0.27 nM. Atropine, but not hexamethonium (a nicotinic antagonist), was able to displace [3H]QNB from IMCD cells with a Ki of 0.1 microM. It is, therefore, concluded that specific high affinity muscarinic receptors are present in IMCD cells. These receptors may play a role in producing the pharmacologic actions of cholinergic agents on the kidney.

Animals↗
PubMed1988

Increased expression of alpha 1-adrenergic receptors in the hypothalamus of spontaneously hypertensive rats.

The specificity and molecular weights of alpha 1-adrenergic receptors in various tissues of spontaneously hypertensive (SH) rat were compared with normotensive controls (Wistar-Kyoto; WKY) with the use of [125I]HEAT and [125I]azidoprazosin, specific alpha 1-adrenergic receptor antagonists. Binding of [125I]HEAT to membranes prepared from SH rat brain hypothalamus was significantly higher, due to a 75% increase in the Bmax, than the WKY control. In contrast, the Bmax and Kd of [125I]HEAT binding to brainstem and liver membranes from SH rats were not significantly different from those of WKY controls. Competition-inhibition data suggested similar pharmacological specificity with potencies in the order of prazosin greater than yohimbine greater than propranolol for both WKY and SH rat membranes prepared from liver, hypothalamus, brainstem and neuronal cultures. Photoaffinity labeling of alpha 1-adrenergic receptors from hypothalamus, brainstem and neuronal cultures using [125I]azidoprazosin followed by SDS-PAGE and autoradiography showed the presence of one major band with a molecular weight (MW) of 105,000 Da for both WKY and SH rats. In contrast, labeling of liver alpha 1-adrenergic receptors revealed one major band with a MW of 60,000 Da. Quantitation of the 105,000-Da band from SH rat hypothalamic membranes demonstrated a 52% higher intensity compared with WKY controls. Neuronal cultures prepared from 1-day-old SH rats showed a similarly greater intensity of the 105,000-Da band compared with WKY controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Receptors for phorbol esters are primarily localized in neurons: comparison of neuronal and glial cultures.

Binding of [3H]PDB has been measured in the present study to determine the levels of protein kinase C in the neuronal and astrocytic glial cells in culture from rat brain. Binding of [3H]PDB to homogenates of cultured neuronal cells from the brains of normotensive and hypertensive rats was time-dependent and specific. The relative potency for competition by various phorbol esters to [3H]PDB binding was TPA greater than beta-PDD greater than POE greater than alpha-PDD greater than or equal to 4 alpha phorbol. Scatchard analysis showed that neuronal cultures from normotensive rat brains contained 2-3 fold more phorbol ester receptors compared with the glial cultures from the same brains. No differences in the Kd and Bmax were observed between neuronal cultures from normotensive and spontaneously hypertensive rat brains. These studies suggest that the phorbol ester receptors are primarily localized in neuronal cells.

Animals↗
PubMed1988

Differential regulation of phosphoinositide phosphodiesterase activity in brain membranes by guanine nucleotides and calcium.

We have shown previously that calcium and guanine nucleotides stimulate the activity of a phosphoinositide (PI) phosphodiesterase in membranes from rat cerebral cortex and that their effects are additive. To understand further guanine nucleotide- and calcium-stimulated PI phosphodiesterase activity, we have investigated the pH sensitivity and effects of inhibitors on the two modes of stimulation. NaF stimulates PI hydrolysis in brain membranes with an EC50 of 2 mM and a maximal effect at 10 mM, suggesting that a guanine nucleotide binding protein can regulate PI phosphodiesterase. Neomycin inhibited guanylylimidodiphosphate (GppNHp)-stimulated PI phosphodiesterase activity in a concentration-dependent manner, with 90% inhibition at 0.3 mM. Neomycin was not as effective at inhibiting calcium-dependent PI hydrolysis (32% inhibition at 0.3 mM). Chloroquine also had a greater inhibitory effect against GppNHp-stimulated PI phosphodiesterase activity compared to calcium-dependent activity. Guanine nucleotide- and NaF-dependent activations of PI phosphodiesterase were strongly pH-dependent, with greatest stimulation observed at pH 5-6 and inhibition at more alkaline pH. Calcium-stimulated PI hydrolysis was not as sensitive to changes in pH and had a peak of activity at pH 9. Our findings of different pH optima and differential sensitivity to inhibitors suggest that calcium and guanine nucleotides may regulate PI phosphodiesterase in rat cortical membranes through independent mechanisms.

Animals↗
PubMed1988

Phorbol ester-induced upregulation of angiotensin II receptors in neuronal cultures is potentiated by a calcium ionophore.

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.

Angiotensin II↗
PubMed1988

Effects of ethanol in vivo and in vitro on stimulated phosphoinositide hydrolysis in rat cortex and cerebellum.

The effects of ethanol on phosphoinositide (PI) hydrolysis in rat cortex and cerebellum were studied to determine if this signal transduction mechanism is a pharmacological site of action for ethanol. In addition PI responses in young adult (8 months old) and old (22 months old) rats were compared to investigate the possible interaction between chronic ethanol treatment and aging on stimulated inositide metabolism. Fischer 344 rats were maintained on a nutritionally complete liquid diet containing sucrose or ethanol for 5 months. PI hydrolysis in prelabeled cortical or cerebellar slices was determined by measuring the release of [3H]inositol phosphates in the presence of 8 mM LiCl. Neither chronic ethanol nor aging altered maximal PI responses to carbachol or submaximal responses elicited by 20 mM KCl or 30 microM A23187. The glutamate-induced response was slightly reduced in the aged rats. Concentration-effect curves for norepinephrine (NE)-stimulated PI hydrolysis were similar in sucrose- and ethanol-treated cortex and cerebellum. Ethanol in vitro inhibited NE-stimulated PI hydrolysis in cortical but not cerebellar slices. The ethanol-induced inhibition of the NE-stimulated PI response was not altered by aging or chronic ethanol treatment. These results suggest that aging or chronic ethanol treatment do not cause large changes in the responsiveness of most PI-linked receptors, and thus, any deficits caused by these conditions may not be due to functional changes in receptor-mediated PI hydrolysis.

Age Factors↗
PubMed1988

Alpha 1-adrenergic receptor stimulated responses.

alpha 1 Receptors have been investigated using a variety of experimental approaches. alpha 1-Receptor stimulation of phosphoinositide (PI) hydrolysis shows differences in agonist efficacy. The potency series and antagonists clearly suggest that alpha 1 receptors are coupled to phosphoinositide hydrolysis. This coupling appears to be mediated by a guanine nucleotide protein coupling the agonist-receptor complex to the phosphoinositide phosphodiesterase. However, certain alpha 1-agonists stimulate phosphoinositide hydrolysis only at very high concentrations that are not sensitive to prazosin antagonism. Other studies on alpha 1-receptor desensitization note discrepancies in coupling to phosphoinositide hydrolysis that are also found when comparing hypertensive (SHR) and normotensive (WKY) rats. Furthermore, molecular studies indicate that the apparent molecular weight of the alpha 1 receptors varies among tissues. These studies suggest heterogeneity in alpha 1 receptors and alpha 1-receptor-mediated responses. This heterogeneity is supported by studies on alpha 1-adrenergic-receptor-mediated decreases in angiotensin II receptors. The response to alpha 1 stimulation is opposite to that found with phorbol esters, which mimic the second messenger response to PI hydrolysis. Thus, these studies suggest that alpha 1-adrenergic receptors are coupled to multiple second messenger responses, one of which is phosphoinositide hydrolysis.

Animals↗
PubMed1988

Cholinergic stimulation of phosphoinositide hydrolysis in rabbit kidney.

An injection of acetylcholine (ACh) into renal artery is known to cause diuresis. In brain and other organs, cholinergic agents have been shown to produce their actions through the phosphoinositide (PI) second messenger system. To determine if cholinergic agents also produce activation of the PI messenger system in the kidney, we investigated the effects of carbachol (a stable analog of acetylcholine) on PI hydrolysis in the cortex, outer medulla and inner medulla of the rabbit kidney. PI hydrolysis was determined by measuring the formation of inositol phosphates in response to stimulation by carbachol in the presence of 8 mM lithium. Carbachol, 1 mM, was able to stimulate PI hydrolysis in the inner medulla and outer medulla (622 and 388% over control values, respectively), but not the cortex. The response to carbachol in the inner medulla was concentration-dependent (EC50 = 10(-5) M). The response was blocked by 1 microM atropine and not by 1 microM hexamethonium. The nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium iodide did not stimulate PI hydrolysis. The effect of carbachol was dependent upon the presence of calcium ions. Substitution of alpha-ketoglutarate for glucose inhibited the response to carbachol in the inner medulla, suggesting a specific substrate requirement in PI metabolism. It is concluded that cholinergic agents produce stimulation of PI hydrolysis through muscarinic receptors in the inner medulla. Whether PI second messenger system in the kidney is involved in the diuretic effect of cholinergic agents remains to be determined.

Animals↗
PubMed1987

Phorbol esters inhibit agonist-stimulated phosphoinositide hydrolysis in neuronal primary cultures.

The effects of phorbol esters on neurotransmitter-stimulated phosphoinositide (PI) hydrolysis in neurons in primary culture were investigated. Ten-day-old neuronal cultures were incubated with [3H]inositol for 2-3 days, exposed to phorbol esters, and the release of [3H]inositol phosphates was measured in the presence of 10 mM lithium. Pretreatment of the neuronal cultures with 1 microM phorbol myristate acetate (PMA) inhibited alpha 1, muscarinic, and glutamate receptor-mediated PI hydrolysis in a time-dependent manner with maximal inhibition observed after a 20-30 min preincubation. The active beta-phorbol didecanoate inhibited stimulated PI hydrolysis, but its stereo-isomer alpha-phorbol didecanoate was without effect at 1 microM. PMA was about 10 times more potent at inhibiting PI hydrolysis stimulated by norepinephrine and glutamate compared to carbachol. The order of potency of the various phorbol esters for inhibition of stimulated PI hydrolysis and the differences between active and inactive stereoisomers suggests that the activation of protein kinase C may mediate the inhibitory effects. Thus, stimulation of neuronal protein kinase C may represent a mechanism for the regulation of agonist-stimulated PI hydrolysis.

Animals↗
PubMed1987

Distinct angiotensin II receptor in primary cultures of glial cells from rat brain.

Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Ang-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125I-Ang-II binding was as follows: Ang-II = [sarcosine1,Ala8]Ang-II greater than [sarcosine1,Ile8]Ang-II much greater than Ang-III greater than Ang-I. Scatchard analysis revealed a homogeneous population of high-affinity (Kd = 1.1 nM) binding sites with a Bmax of 110 fmol/mg of protein. Light-microscopic autoradiography of 125I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [3H]norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors.

Angiotensin II↗
PubMed1987

Variations in membrane sensitivity of brain region synaptosomes to the effects of ethanol in vitro and chronic in vivo treatment.

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.

Animals↗
PubMed1987

Protein kinase C agonists increase the expression of angiotensin II receptors in neuronal cultures.

Previous studies have shown that norepinephrine is important in the regulation of central angiotensin II receptors, an effect mediated by alpha 1-adrenergic receptors. Because alpha 1-adrenergic stimulation leads to inositol phospholipid hydrolysis and activation of protein kinase C, we have examined a possible role of this enzyme in the regulation of central angiotensin II (Ang II) receptors. In the present study, we have examined the effects of protein kinase C activators, phorbol esters, on the expression of Ang II receptors in neuronal cultures prepared from 1-day-old rat brains. The active phorbol ester phorbol-12-myristate-13-acetate (TPA) caused time- and concentration-dependent increases in the specific binding of [125I]Ang II to its receptors in neuronal cultures of normotensive and spontaneously hypertensive rat brains. The stimulatory effect of TPA on Ang II receptors was apparent within 15 min and reached a maximum between 1 and 2 h. Ang II specific binding had returned to control levels by 24 h. Various phorbol esters increased [125I]Ang II binding in accordance with their order of potency in stimulating protein kinase C activity. Saturation and Scatchard analysis revealed that the phorbol ester-induced increase in [125I]Ang II binding was due to an increase in the number of Ang II receptors. These observations indicate that activation of protein kinase C results in an increased expression of Ang II receptors in neuronal cultures from both normotensive and spontaneously hypertensive rat brains. The results suggest a possible role of phosphorylation in Ang II receptor expression in neuronal cultures.

Angiotensin II↗
PubMed1987

Norepinephrine regulation of alpha-1 receptors and alpha-1-stimulated phosphoinositide hydrolysis in primary neuronal cultures.

Alpha-1 receptor density and alpha-1 receptor-stimulated phosphoinositide hydrolysis in neuronal primary cultures are regulated by exposure of the cells to the alpha-1 agonist norepinephrine (NE). Pretreatment of neuronal cultures with 10 microM NE caused a time- and concentration-dependent decrease in NE-stimulated accumulation of [3H]inositol phosphates. The maximal NE-stimulated inositide hydrolysis was decreased by approximately 80% after 2 hr of pretreatment with NE, and this loss of responsiveness was reversible over a period of 12 hr. NE pretreatment of neuronal cultures did not affect the muscarinic receptor stimulation of inositide hydrolysis. Neither the number of alpha-1 receptor recognition sites, assessed by measuring the specific binding of DL-[125I]-alpha-(3-iodohydroxyphenyl)-ethyl-aminomethyl tetralone ([125I]HEAT), nor the Ki for NE inhibition of [125I]HEAT binding were changed appreciably after treating neurons with NE for 2 hr. A time- and concentration-dependent decrease in alpha-1 receptor recognition sites occurred with longer periods of NE pretreatment. The maximal loss of 50 to 60% [125I]HEAT binding sites was seen after 14 hr of pretreatment with NE. The number of [125I]HEAT binding sites returned to control levels within 24 hr in parallel with the recovery of alpha-1-stimulated inositide hydrolysis after being down-regulated by a 24-hr exposure to NE. Cycloheximide (3.5 microM) blocked both the recovery of alpha-1 receptors and the recovery of alpha-1-stimulated inositide hydrolysis after 24 hr of NE pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Ethanol effects on receptor activated lipid hydrolysis in brain and liver.

The effects of ethanol in receptor-stimulated phosphoinositide (PI) hydrolysis in brain and liver slices were investigated. Norepinephrine (NE) was used as an alpha 1-adrenergic receptor agonist to stimulate PI hydrolysis in both tissues. Stimulation of PI hydrolysis by maximal concentrations of norepinephrine (e.g., 100 microM) was inhibited approximately 50% by 500 mM in vitro ethanol in slices from the cerebral cortex, hippocampus, hypothalamus, and striatum. NE-stimulated PI hydrolysis was not affected by 500 mM ethanol in brain stem slices. KCl (20 mM)-stimulated PI hydrolysis was strongly inhibited in all of the above brain regions in the presence of 500 mM ethanol. To further characterize the effects of ethanol on PI hydrolysis in brain, an isolated membrane preparation was used. Guanine nucleotide and calcium-dependent PI hydrolysis in cortical membranes was not affected by concentrations of ethanol as high as 500 mM. In liver slices, NE-stimulated PI hydrolysis was inhibited 50% by 30 mM ethanol in vitro. These results suggest that ethanol may interfere with the coupling of the alpha 1 receptor to the phosphoinositide phosphodiesterase in brain and liver.

Animals↗