Receptors that inhibit phosphoinositide breakdown.

Calcium-mobilizing receptors are believed to activate phospholipase C. Joel Linden and Thérèse Mary Delahunty summarize recent reports which indicate that activation of some receptors that inhibit the accumulation of Ca2+ within cells - notably receptors for adenosine, dopamine and several other neurotransmitters - can inhibit phosphoinositide metabolism. Two types of mechanism may be involved in these responses. Many instances of receptor-mediated inhibition of phosphoinositide breakdown can be detected only after a period of several minutes and may be secondary to receptor-mediated events that lower [Ca2+]i or activate certain protein kinases. In other instances the activation of receptors rapidly (within seconds) inhibits phosphoinositide breakdown, possibly via the activation of guanine nucleotide binding proteins that either directly, or by a rapid indirect action, inhibit phospholipase C. Putative mechanisms for direct and indirect regulation of phospholipase C are discussed.

The hydroxylamine of sulfamethoxazole and adverse reactions in patients with acquired immunodeficiency syndrome.

We measured the urine concentrations of sulfamethoxazole, sulfamethoxazole hydroxylamine, and N-sulfamethoxazole on days 3 and 10 in 15 patients with acquired immunodeficiency syndrome treated with a combination product of trimethoprim (15 mg/kg/day) and sulfamethoxazole (75 mg/kg/day). The percentage of sulfamethoxazole and metabolites excreted on days 3 and 10, respectively, were sulfamethoxazole 17.2% +/- 11.3% versus 15.6% +/- 8.2%; sulfamethoxazole hydroxylamine 2.6% +/- 2.0% versus 5.0% +/- 5.2% (p < 0.05); N-acetylsulfamethoxazole 80.0% +/- 12.9% versus 79.8% +/- 11.8%. The percentage of sulfamethoxazole hydroxylamine excreted was similar between the eight patients who discontinued therapy because of toxicity and the seven patients who did not (2.9% +/- 2.3% versus 2.3% +/- 2.0%, p = 0.7). In two patients who had major liver toxicity the percentage of sulfamethoxazole hydroxylamine excreted was significantly lower than that of the 13 patients who did not (0.8% +/- 0.1% versus 2.9% +/- 2.0%, p < 0.05). This is the first report of the formation and excretion of sulfamethoxazole hydroxylamine in patients with acquired immunodeficiency syndrome. With 15 patients we were unable to show a significant correlation between the percentage of sulfamethoxazole hydroxylamine excreted and adverse reactions. However, patients with liver toxicity excreted less sulfamethoxazole hydroxylamine.

Maitotoxin increases inositol phosphates in rat anterior pituitary cells.

Maitotoxin is a potent marine poison that mobilizes calcium in most vertebrate cell types and accelerates secretion from anterior pituitary cells. It is not known whether voltage-sensitive calcium channels or other mechanisms initiate the effects of maitotoxin on anterior pituitary cells. Changes in intracellular Ca2+ levels may also be achieved by releasing internal calcium stores via inositol trisphosphate (InsP3). Indeed, maitotoxin rapidly increased inositol phosphate accumulation in a concentration-dependent manner. Calcium channel antagonists such as nifedipine and verapamil did not block this response nor did calcium-mobilizing agents (BAYk8644, A23187) mimic this effect. These data suggest that the mechanism by which maitotoxin acts at the pituitary may include the activation of an enzyme that produces the calcium-mobilizing signal InsP3.

Differential consequences of lateral and central fluid percussion brain injury on receptor coupling in rat hippocampus.

We have identified alterations in the responses of muscarinic and metabotropic receptors in rat hippocampus that persist for at least 15 days after central fluid percussion injury. This study compares the effect of lateral fluid percussion and central fluid percussion on these responses. Moderate injury was obtained by displacement and deformation of the brain within the closed cranial cavity using a fluid percussion device positioned either centrally or laterally. Carbachol and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated polyphosphoinositide (PPI) hydrolysis was assayed in hippocampus from injured and sham-injured controls at 15 days following injury. At 15 days after central fluid percussion traumatic brain injury (TBI), the response to carbachol was enhanced by 30% and the response to trans-ACPD was enhanced by 75% compared to sham-injured animals. At 15 days after lateral fluid percussion TBI the response to trans-ACPD was enhanced by 40% both ipsilateral and contralateral to the side of injury. In contrast, the response to carbachol was enhanced by 29% contralateral to the side of injury but was diminished by 12% ipsilateral to the side of injury. Cresyl violet staining shows no hippocampal cell death after central fluid percussion injury or on the side contralateral to lateral fluid percussion injury but on the ipsilateral side cell death was identified in hippocampal area CA3. Thus, abnormal hippocampal cell signaling through the phosphoinositide pathway occurs in the absence of cell death and may contribute to cognitive impairment.

Subtle alterations in NMDA-stimulated cyclic GMP levels following lateral fluid percussion brain injury.

This study examined whether NMDA-stimulated cyclic GMP levels were altered at two different time points following lateral fluid percussion injury. At 60 min and 15 days postinjury, the left and right hippocampi were dissected and chopped into mini-prisms. Each hippocampus was divided into five equal parts and incubated with either the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine, 500 microM) alone, IBMX and N-methyl-D-aspartic acid (NMDA) OR IBMX, NMDA, and glycine (10 MM). Two concentrations of NMDA were used: 500 or 1,000 microM. Tissues were then assayed for levels of cyclic GMP. Results indicated that there were no changes in basal levels of cyclic GMP at either postinjury time point. At 60 min postinjury, there were no significant main effects for injury or drug concentration. There was a significant injury x side interaction effect with increased levels of NMDA-stimulated cyclic GMP in the hippocampus ipsilateral to the injury impact and decreased cyclic GMP levels in the contralateral hippocampus. There were no significant alterations in NMDA-stimulated cyclic GMP levels at 15 days postinjury. The data from this study indicated that NMDA-stimulated cyclic GMP accumulation is differentially altered in the hippocampus ipsilateral and contralateral to the site of the injury at 1 h after injury, but is normalized by 15 days postinjury. These findings implicate NMDA-mediated intracellular signaling processes in the acute excitotoxic response to injury.

Mild traumatic brain injury enhances muscarinic receptor-linked inositol phosphate production in rat hippocampus.

Recent evidence suggests that muscarinic receptors play a role in the hippocampal hypersensitivity to imposed ischemia following mild traumatic brain injury (TBI). In rat hippocampal tissue, carbachol-stimulated inositol phosphate production was found to be enhanced by mild TBI, at 1 h post injury. This finding suggests that mild TBI may result in enhanced coupling between muscarinic receptors and phosphoinositide hydrolysis, which may contribute to post-traumatic hippocampal vulnerability to secondary ischemia.

Muscarinic cholinergic receptor binding in rat brain following traumatic brain injury.

Recent evidence suggests that excessive activation of muscarinic cholinergic receptors (mAChRs) contributes significantly to the pathophysiological consequences of traumatic brain injury (TBI). To examine possible alterations in mAChRs after TBI, the affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, brain stem and cerebellum were determined by [3H]QNB binding. Three groups of rats were examined: 1 h post-TBI (n = 21), 24 h post-TBI (n = 21) and sham-injured rats (n = 21). Kd values were significantly higher in hippocampus and brain stem at 1 but not 24 h post-TBI compared with sham-injured controls (P < 0.05). Kd values did not significantly differ in neocortex and cerebellum at 1 or 24 h post-TBI compared with sham-injured controls. Bmax values did not significantly differ in any brain areas at 1 or 24 h post-TBI compared with sham-injured controls. These results show that TBI significantly decreases the affinity of mAChRs in hippocampus and brain stem at an early stage post-TBI, which may contribute to desensitization of mAChRs after TBI. The findings of no change in Bmax values are consistent with a transient elevation in ACh concentrations after TBI.

Muscarinic cholinergic receptor binding in rat brain at 15 days following traumatic brain injury.

Laboratory studies indicate that activation of muscarinic cholinergic receptors (mAChRs) at or soon after traumatic brain injury (TBI) significantly contributes to behavioral morbidity. Recent research has demonstrated that pre-injury treatment with the muscarinic antagonist scopolamine significantly reduces spatial memory deficits at 11-15 days post-TBI. In the present study, we examined mAChR binding kinetics in brain regions at 15 days after moderate (1.95 atm) fluid percussion TBI in untreated and scopolamine-treated rats. Three groups were examined: untreated TBI (n = 8), TBI with pre-injury scopolamine treatment (1.0 mg/kg, i.p., 15 min prior to injury) (n = 11), and sham-injury (n = 7). The affinity (Kd) and maximum number of binding sites (Bmax) of mAChRs in hippocampus, neocortex, and brainstem were determined by [3H]QNB binding. Bmax values in TBI animals were significantly higher in hippocampus (4061 +/- 494 fmol/mg protein) and neocortex (4272 +/- 640 fmol/mg protein), but not in brainstem (833 +/- 39 fmol/mg protein) compared to sham-injured controls (hipp. 2812 +/- 218 fmol/mg/protein; neoctx. 2850 +/- 129 fmol/mg protein; brainstem 794 +/- 26 fmol/mg protein) (P < 0.05). At 15 days after injury, Bmax values of mAChRs in TBI animals with pre-injury scopolamine treatment (hipp. 2850 +/- 129 fmol/mg protein; neoctx. 2948 +/- 123 fmol/mg protein) did not differ from control. In all brain regions, Kd values did not differ between groups. These results demonstrate that TBI significantly alters the binding sites of mAChRs in hippocampus and neocortex for as long as 15 days after TBI. Furthermore, these results indicate that a pharmacological treatment that improves motor and memory function outcome also normalizes aspects of mAChRs physiology. These data suggest that excessive activation of mAChRs at or soon after TBI impact contributes to long-term pathophysiological processes in TBI.

Metabotropic glutamate antagonist, MCPG, treatment of traumatic brain injury in rats.

The metabotropic glutamate receptor (mGluR) antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) was administered into the left lateral ventricle 5 min prior to fluid percussion traumatic brain injury (TBI) in the rat. A single 5.0 microliters ventricular infusion of the active isomer. (+)-MCPG (0.2 mumol), significantly reduced beam walking motor deficits on days 1-5 after injury and learning/memory deficits measured on days 11-15 after injury. Neither a lower dose of (+)-MCPG (0.2 mumol) affected behavioral outcome. (+)-MCPG (0.2 mumol) did not affect systemic hemodynamic responses to injury. These results suggest that TBI induced activation of mGluRs contributes to behavioral morbidity and that blockade of certain mGluR subtypes (mGluR1, mGluR5 and/or mGluR2) may reduce these pathophysiological responses.

A study of endogenous N-cholylglycine distribution among serum proteins using radioimmunoassay.

The amount of N-cholylglycine endogenously associated with the various protein fractions was assessed in serum of patients with hepatobiliary obstruction using a sensitive radioimmunoassay. In contrast to evidence obtained with cholate, the albumin fraction separated by electrophoresis was found to contain only 2.6% of the total N-cholylglycine, while the alpha 2 region had 64% associated with it. The serum beta-lipoprotein isolated by anionic precipitation also contained significant quantities of the conjugated bile acid, the amount in this fraction being greater in the serum of hyperlipidemic subjects. The results suggest that despite accounting for some 63% of the total serum protein, albumin is not the major carrier of endogenous N-cholylglycine in the circulation.

Toxic effect on the rat small intestine of chronic administration of asbestos in drinking water.

Sprague-Dawley rats were given a 0.5 g/l Chrysotile asbestos solution in their drinking water (approximately 7 mg/day ingested) for 1.5 years and compared to control rats of the same age. During this time there were no differences in weight or appearance of the asbestos-treated rats in comparison to controls maintained under the same conditions. However, when in vivo intestinal permeability studies were performed using a gavage/urinary collection technique, some significant changes were noted. The recovery of lactulose in the urine of asbestos-treated rats was 0.66 +/- 0.07%, significantly less than that of the controls (1.01 +/- 0.08, P less than 0.005). The recovery of mannitol was similarly decreased (2.2 +/- 0.28 vs. 3.0 +/- 0.17, P less than 0.02), but that of rhamnose was unchanged. Creatinine clearance studies indicated that there was no impairment of kidney function in the asbestos-treated group and polarized light microscopy did not reveal any asbestos fibers in sections of the small bowel. The results suggest that the chronic exposure of rats to asbestos fibers in the drinking water results in a decreased ability of the intestine to absorb some non-metabolizable sugars.

Intestinal permeability changes in rodents: a possible mechanism for degraded carrageenan-induced colitis.

Rats and guinea-pigs were treated with degraded carrageenan (50 g/litre in the drinking-water) and their intestinal permeability was studied at weekly intervals over the last 4 wk of the test period by determining the recovery of orally administered tracer doses of [3H]polyethylene glycol (PEG-900) or D-[3H]mannitol in 16-hr urine collections. A freely diffusible dye, Azure A, was administered simultaneously to compensate for non-intestinal factors that could modify renal excretion. Animals were killed after a total treatment period of 5 months for rats and 6 wk for guinea-pigs. After 3 wk of carrageenan treatment, excretion of PEG-900 (expressed as a ratio of the Azure A excretion) in guinea-pigs showed a statistically significant increase over that in the control group. At autopsy, the caeca showed numerous macroscopically visible erosions of the entire mucosal surface and histological examination showed ulcerations largely in the mucosa with abscesses in the crypts. Although no such histological changes were seen in the intestines of the treated rats, even after 5 months, a statistically significant increase in PEG-900 excretion was again found compared with the control group. This increase did not occur when deoxycholate was administered with the carrageenan solution. No effect of carrageenan treatment on mucosal permeability to D-[3H]mannitol was demonstrated in either species. The results suggest that degraded carrageenan-induced colitis could be a result of increased intestinal permeability, since ingestion of this polysaccharide by rats increased PEG-900 absorption without causing mucosal damage.

Human retinal pigment epithelial cells possess V1 vasopressin receptors.

Membrane preparations of cultured human retinal pigment epithelial (RPE) cells were incubated with various concentrations of [3H]arginine vasopressin (AVP) in the presence and absence of 10 microM nonradioactive AVP. Saturable, specific binding to a single site with a Kd of 6.2 nM and Bmax of 111 fmol/mg protein was detected. Vasopressin had no effect on RPE cyclic AMP levels measured by radioimmunoassay. Intracellular calcium fluxes were measured by spectrofluorometry of RPE cell suspensions preloaded with quin 2. The baseline cytosolic calcium level was 217 +/- 20 nM, and AVP caused a concentration-dependent increase in this level with a 3.5-fold maximal response at 10(-6) M and an EC50 of 120 nM. The production of inositol phosphates was measured in RPE preloaded with [3H]myoinositol, and AVP caused a concentration-dependent increase in their production with a 2.1-fold maximal response at 10(-5) M and an EC50 of 80 nM. A specific vasopressin receptor antagonist, SKF 101926, prevented the AVP-induced increase in calcium mobilization and inositol phosphate production in RPE. These data suggest that RPE cells possess V1 AVP receptors coupled to calcium mobilization and inositol phosphate metabolism.

Caffeine demethylation monitoring using a transdermal sweat patch.

Caffeine and two metabolites (paraxanthine and theobromine) were quantitated by high-performance liquid chromatography (HPLC) using extracts from transdermal sweat patches that continuously collected and stored analytes lost through the skin. Following caffeine consumption, remarkably clean chromatograms were obtained with minimal sample preparation. Caffeine and paraxanthine accumulated in the patch at comparable rates, and theobromine accumulated more slowly. A major urinary metabolite, 1-methylxanthine, was notably absent in sweat-patch and plasma extracts, a finding which favors a renal source for this metabolite. A simple, noninvasive approximation of N-demethylation can be made by calculating the paraxanthine/ caffeine and theobromine/caffeine ratios in the patch extract. These ratios were significantly reduced in high-dose (600 mg) versus low-dose (200 mg) subjects, possibly reflecting a decreased clearance of caffeine. Because the sweat patches can be worn for several days, the technique gives a multiday historical record which reflects the fluctuating systemic concentration of caffeine and its hepatic metabolites and thus might be useful to noninvasively monitor compliance by caffeine-restricted patients or to assess drug-metabolizing status.

The effect of lopinavir/ritonavir on the renal clearance of tenofovir in HIV-infected patients.

We determined the effects of lopinavir/ritonavir on tenofovir renal clearance. Human immunodeficiency virus-infected subjects taking tenofovir disoproxil fumarate (TDF) were matched on age, race, and gender and were enrolled into one of the following two groups: group 1: subjects taking TDF plus lopinavir/ritonavir plus other nucleoside reverse transcriptase inhibitors (NRTIs); group 2: subjects taking TDF plus NRTIs and/or non-NRTIs but no protease inhibitors. Twenty-four-hour blood and urine collections were carried out in subjects for tenofovir quantification. Drug transporter genotype associations with tenofovir pharmacokinetics were examined. In 30 subjects, median (range) tenofovir apparent oral clearance, renal clearance, and fraction excreted in urine were 34.6 l/h (20.6-89.5), 11.3 l/h (6.2-22.6), and 0.33 (0.23-0.5), respectively. After adjusting for renal function, tenofovir renal clearance was 17.5% slower (P=0.04) in subjects taking lopinavir/ritonavir versus those not taking a protease inhibitor, consistent with a renal interaction between these drugs. Future studies should clarify the exact mechanism and whether there is an increased risk of nephrotoxicity.

Convenient screening for hemoglobin variants by using the Diamat HPLC system.

Hemolysates from 102 different patients previously assessed for the presence of hemoglobin variants by cellulose acetate electrophoresis were reanalyzed with the Diamat, a microprocessor-controlled step-gradient HPLC technique designed to determine glycohemoglobin (Hgb A1c). This pool contained 81 abnormal specimens, each with one of seven different variant abnormalities. In all cases the HPLC technique correctly detected the presence or absence of a variant. The common S trait gave a large peak immediately after Hgb A, and the SS and SC variants gave clearly abnormal patterns, caused in part by the absence of Hgb A. The pattern from EE variants was distinguished by the appearance of the glycated fraction Hgb E1c, which is eluted at 4.7 instead of 4.2 min, whereas the AE pattern had two glycated peaks. Hemoglobins F, "fast", S, and C were discerned by the presence of a major peak at 3, 5.2, 6.5, and 7.5 min, respectively. The findings suggest that the Diamat is a convenient tool to detect and help diagnose variants in a screening pool.

Creatine kinase-BB isoenzyme in rat plasma after chronic lead intoxication.

Creatine kinase (CK) activity in plasma obtained non-invasively from adult healthy, Sprague-Dawley, male rats was found to be 528 +/- 270 U/L (N = 17), a value which was 7 times that obtained in human specimens. Agarose gel electrophoresis revealed that the only detectable CK isoenzyme present was CK-BB, in contrast to the human serum isoenzyme which was CK-MM. Furthermore, it was found that the rat CK-BB could be detected using an RIA technique designed to quantitate human CK-BB occasionally present in blood after brain injury (rat CK-BB = 84.5 +/- 55.2 micrograms/L, N = 17, human CK-BB: Not detectable). It was thus possible to calculate the CK-BB specific activity (SA) in rat plasma using total CK assay and RIA (rat CK-BB SA = 6.25 +/- 3.87 U/micrograms, N = 17). When six rats (156 +/- 23 g) were treated with lead acetate in the drinking water (26 mM) for 3 weeks, the CK-BB SA rose to 18 +/- 5.8 U/micrograms (P less than .02). At this point the electrophoresis pattern of the CK-BB showed a transient change from a single band to a doublet. The dose was then increased to 52 mM for 6 weeks, during which time the CK-BB SA declined steadily to 1.6 +/- 0.6, a level significantly less than that of the untreated animals (p less than .02). The results suggest that chronic lead treatment evokes a biphasic response in CK-BB SA with the initial release of enzyme of high SA from tissues. Further treatment apparently results in an inactivation of the enzyme within lead sensitive tissues.

A new technique for studying the relationship between maternal diabetes and the sialic acid content of fetal pulmonary surfactant.

By adapting a standard method for precipitation of high-density lipoprotein cholesterol with phosphotungstic acid (PTA) and Mg2+, fetal pulmonary surfactant can be rapidly isolated from human amniotic fluid, 97% of the total disaturated phosphatidylcholine being precipitated from the sample. The lecithin/sphingomyelin ratio for 17 separate specimens correlated reasonably well (r = 0.76) with the concentration of disaturated phosphatidylcholine in the PTA precipitate. Using thiobarbituric acid as the chromophore, I measured sialic acid in the PTA precipitate after overnight treatment with neuraminidase. The sialic acid/protein ratio for the PTA precipitate was identical to that for the surfactant, as isolated by ultracentrifugation. The concentrations of insulin and C-peptide were significantly greater in specimens of amniotic fluid from mothers with diabetes than from non-diabetic mothers (p less than 0.001). When the specimens were segregated according to a C-peptide cutoff value of 4 micrograms/L, there was a small, significant decrease in PTA-precipitated concentrations of sialic acid in the samples with C-peptide greater than 4 micrograms/L. The results suggest a possible mechanism for the increased incidence of respiratory distress among infants born to diabetic mothers.

Cathodally migrating creatine kinase in sera of patients treated with theophylline, and its diagnostic implications.

Sera from 30 patients, selected only on the basis of having serum theophylline concentrations greater than or equal to 18 mg/L, were assayed for total creatine kinase (CK), isoenzyme CK-MB, and cathodally migrating CK (mCK-2). CK-MB was measured by ion-exchange chromatography. mCK-2 was detected by electrophoresis on agarose gel, with fluorometric scanning. The mCK-2 variant was found in 37% of these 30 patients. None of the patients in the mCK-2 group had CK-MB values exceeding the cutoff value associated with myocardial infarction, so extensive cardiac ischemia was considered unlikely. A review of the patients' records supported this conclusion, and no particular association with any specific disease was found among the mCK-2 group. Investigations with use of goat anti-CK-MM and anti-theophylline indicated that neither CK-MM nor theophylline were present in the mCK-2. Results of specific radioimmunoassay for CK-B subunit were negative. The mobility of the mCK-2 peak was unresponsive to 2-mercaptoethanol, suggesting that CK of mitochondrial origin was not present. These results provide insight into the clinical relevance of mCK-2 and some information concerning its composition.