Interactions in indices of vitamin A, zinc and copper status when these nutrients are fed to rats at adequate and increased levels.

The purpose of the present study was to determine the effects of feeding nutritionally adequate and increased levels of vitamin A (retinyl acetate at 1.4, 34.4, and 206.4 mg/kg diet) in combination with adequate or increased Zn (12 and 240 mg/kg) and Cu (5 and 50 mg/kg) on serum and tissue concentrations of retinol and retinyl palmitate and on indices of Cu and Zn status in female Sprague-Dawley rats, and to measure interactive effects of such nutrient imbalances. Rats fed on diets containing 34.4 and 206.4 mg vitamin A/kg had higher feed intakes and relative liver weights than those fed on diets containing 1.4 mg vitamin A/kg. An interaction between dietary Cu and Zn and an independent effect of vitamin A affected serum ceruloplasmin oxidase (EC 1.16.3.1) activity. Rats fed on high Zn, adequate-Cu diets (240 and 5 mg Zn and Cu/kg respectively) had lower serum ceruloplasmin oxidase levels than rats fed on adequate-Zn, adequate-Cu diets (12 and 5 mg Zn and Cu/kg respectively). This effect was not observed in rats fed on high-Zn, high-Cu diets (240 and 50 mg Zn and Cu/kg respectively). Alterations in dietary levels of Cu and vitamin A independently affected haemoglobin levels. Serum cholesterol concentration was affected by interactions between Zn and vitamin A and Cu and vitamin A. Levels of retinol and retinyl palmitate in liver and kidney were significantly higher in rats fed on diets with increased dietary vitamin A than in those fed on diets with adequate vitamin A. Three-way interactions among Cu, Zn, and vitamin A affected levels of retinol in serum and liver. Two-way interactions between Cu and vitamin A affected liver retinyl palmitate and the sum of liver retinol+retinyl palmitate. An independent effect of dietary Zn on these variables was also observed. Interactions between Cu and vitamin A affected levels of Cu in liver and kidney, while Fe and Zn in kidney were affected by interactions between Cu and Zn. This study demonstrates that differing interactions among variables of vitamin A metabolism and mineral status occur with higher dietary levels of vitamin A, Zn and Cu in the rat.

The effects of dietary taurocholate, fat, protein, and carbohydrate on the distribution and fate of dietary beta-carotene in ferrets.

Dietary beta-carotene has been shown to have cancer chemopreventive action on the basis of epidemiologic evidence and studies in animals. Because the anticarcinogenic property of beta-carotene may be exerted per se, it is desirable to achieve the maximum absorption and accumulation of intact beta-carotene in various parts of the body. Therefore the effects of dietary taurocholate, fat, protein, and carbohydrate on the absorption, accumulation, and fate of dietary beta-carotene (3.730 nmol/g diet) in selected tissues of ferrets were explored. Taurocholate (0.2-1.0% wt/wt) and fat (6-23% wt/wt) caused two- to threefold (p < 0.05) increases in the absorption and accumulation of beta-carotene in the liver, lungs, and adipose tissue in a dose-dependent manner. In contrast, neither dietary protein (10-40% wt/wt) nor carbohydrate (25-55% wt/wt) affected the absorption and accumulation of beta-carotene in various tissues. Significantly, taurocholate, 23% fat, or 40% protein also markedly increased the amounts of hepatic retinol and retinyl esters derived from dietary beta-carotene. These results indicate that dietary taurocholate, fat, and high protein have a marked influence on the exposure of beta-carotene to intestinal carotene cleavage enzyme or its activity. Thus an ideal combination of dietary components (wt/wt) in ferrets for the maximal absorption and accumulation of beta-carotene in different tissues is 0.5% taurocholate and 13.4% fat, whereas 1% taurocholate, 23% fat, or 40% protein stimulates its conversion to vitamin A.

Safety and tolerance of intravenous nimodipine.

OBJECTIVE: To evaluate the safety and tolerance of intravenous nimodipine administered via a peripheral vein in healthy male volunteers. DESIGN: Double-blind, placebo- and vehicle-controlled trial with three fixed-dose panels. SETTING: Inpatient infusion and observation periods. SUBJECTS: 61 healthy male volunteers, aged 18-40 years. METHODS: Subjects in panel 1 received nimodipine 0.4 mg/h, vehicle 2 mL/h, or placebo 2 mL/h; subjects in panel 2 received nimodipine 1 mg/h, vehicle 5 mL/h, or placebo 5 mL/h; subjects in panel 3 received nimodipine 2 mg/h, vehicle 10 mL/h, or placebo 10 mL/h. All infusions were administered intravenously for 48 hours and volunteers were observed for 48 hours after cessation of the infusion. In addition to standard safety assessments (physical examination, electrocardiogram, laboratory studies, and adverse event reporting), supine and standing blood pressures and pulse rates were measured frequently. Plasma samples were also analyzed for nimodipine and its two demethylated metabolites. RESULTS: Of 61 subjects, 55 completed the 48-hour infusion and 6 discontinued the study because of adverse events. Intravenous nimodipine was well tolerated at 0.4 and 1 mg/h. However, all six subjects who received nimodipine 2 mg/h experienced moderate-to-severe adverse events, and one subject was discontinued because of dizziness, diaphoresis, and postural hypotension. The matched vehicle (10 mL/h) also was not well tolerated, with three subjects who discontinued because of phlebitis. Two subjects who received placebo were also discontinued during the study. Small (2 mm Hg) decreases in mean supine diastolic blood pressure were observed in the 0.4- and 1-mg/h nimodipine groups, but the 2-mg/h group showed a slight (5 mm Hg) increase in blood pressure. These changes were not clinically significant. Clearance and half-life of nimodipine and its metabolites were similar at all three dosages. CONCLUSIONS: Using peripheral vein administration, nimodipine 2 mg/h and matched vehicle at 10 mL/h were not well tolerated in this healthy normal population. The maximum tolerated dose of nimodipine was found to be 1 mg/h.

Developmental effects of combined exposure to ethanol and vitamin A.

The potential for ethanol (EtOH) to influence the developmental toxicity of vitamin A was investigated. 11 groups of approximately 31 FDA-bred Osborne-Mendel rats received either a control or isocaloric 6.4% EtOH liquid diet (containing 4000 IU vitamin A/litre) ad lib. The vehicle control, EtOH and pair-fed (pair-fed against the EtOH group) groups received corn oil (the vehicle) by gavage. Vitamin A was administered by gavage without EtOH at 40,000, 80,000, 120,000 or 160,000 IU/kg daily. Vitamin A was administered by gavage at 10,000, 20,000, 40,000 or 80,000 IU/kg with EtOH ad lib., daily throughout the study. Combined EtOH and vitamin A resulted in significant reductions in maternal diet consumption and body weight when doses of vitamin A were as low as 10,000 IU/kg. The most severe effects on overall (days 0-20) maternal body weight gain were observed in the groups receiving 120,000 or 160,000 IU vitamin A/kg alone or EtOH in combination with 80,000 IU vitamin A/kg. The overall diet consumption (days 0-20) paralleled the overall weight gain. In general, pups exposed to ethanol and vitamin A had a tendency to weigh less than those exposed to vitamin A alone, but to weigh more than those exposed to EtOH alone. EtOH combined with vitamin A at 80,000 IU/kg resulted in an increased incidence of cleft palate relative to the vehicle control or either treatment alone. The incidence of exencephaly and protruding tongue was significantly greater in the group given vitamin A at 160,000 IU/kg, compared with the vehicle control group. The most consistent statistically significant skeletal finding in the groups receiving combined treatment was a treatment-related increased incidence of supernumerary ribs [14th rib (C7), 14th rib bud (L1) and 15 ribs]. In addition, the incidence of misshapen zygomatic arch was also significantly increased in the group exposed to EtOH and vitamin A at 80,000 IU/kg. The incidence of moderately enlarged renal pelvis and severely enlarged ureter proximal to the kidney was increased in the group exposed to EtOH and vitamin A at 80,000 IU/kg relative to the vehicle control, or either treatment alone. Therefore, for some of the endpoints examined in this investigation, it would appear that ethanol potentiates the developmental effects of vitamin A.

Effect of ethanol and vitamin A excess on vitamin A status in the liver, plasma and foetuses of pregnant rats.

The effect of maternal consumption of dietary ethanol and high doses of vitamin A by gavage was investigated by evaluating plasma, liver and foetal vitamin A in Osborne-Mendel pregnant rats with a view to assessing whether ethanol modulated the potential toxicity of excess vitamin A. All groups received 4000 IU vitamin A/litre in a liquid diet. Ethanol-exposed groups also received 6.4% (v/v) ethanol in the liquid diet. Vitamin A was administered by gavage once per day in corn oil in doses ranging from 10,000 to 160,000 IU/kg body weight. Plasma vitamin A levels in ethanol-exposed groups were similar to levels in a pair-fed group. Plasma vitamin A levels were similar in the group given ethanol plus 40,000 IU vitamin A/kg and the group given 40,000 IU vitamin A/kg only, but were higher in the group receiving ethanol plus 80,000 IU vitamin A/kg than in the group given 80,000 IU vitamin A/kg only. Retinyl esters were present in the plasma of animals receiving 160,000 IU vitamin A/kg only, indicating possible saturation of the liver with vitamin A. Retinyl palmitate levels in female foetuses of the group administered ethanol plus 80,000 IU vitamin A/kg were significantly higher than those of the group administered 80,000 IU vitamin A/kg only; no significant differences in levels of retinyl palmitate in male foetuses were observed between these two groups. This observation suggests a possible sex difference in the modulation of vitamin A toxicity by ethanol in the foetus.

Long-term weight control study. I (weeks 0 to 34). The enhancement of behavior modification, caloric restriction, and exercise by fenfluramine plus phentermine versus placebo.

To investigate the value of anorexiant medications as an adjunct to other forms of weight control therapy, we studied 121 people in a 34-week, double-blind clinical trial of 60 mg extended-release fenfluramine plus 15 mg phentermine resin versus placebo added to behavior modification, caloric restriction, and exercise. Participants weighed 130% to 180% (154% +/- 1.2%, mean +/- SEM) of ideal body weight (1983 Metropolitan Life tables) and were in good health. By week 34, participants receiving active medication lost an average of 14.2 +/- 0.9 kg, or 15.9% +/- 0.9% of initial weight (n = 58), versus a loss of 4.6 +/- 0.8 kg or 4.9% +/- 0.9% of initial weight by subjects taking placebo (n = 54; p less than 0.001). On visual analog scales, participants rated fenfluramine plus phentermine as more helpful than placebo (50.3 +/- 0.5 versus 20.3 +/- 0.3) and not bothersome (fenfluramine plus phentermine, 17.4 +/- 0.3 versus 13.5 +/- 0.2). Blood pressure decreased and pulse remained unchanged in both groups. Dry mouth was the most common adverse effect in subjects receiving fenfluramine plus phentermine; all adverse effects decreased after 4 weeks. Only nine participants left the study in the first 34 weeks. Two subjects from each group left the study as a result of adverse effects. Overall, fenfluramine plus phentermine used in conjunction with behavior modification, caloric restriction, and exercise aided weight loss and continued to be efficacious for 34 weeks.

Long-term weight control study. II (weeks 34 to 104). An open-label study of continuous fenfluramine plus phentermine versus targeted intermittent medication as adjuncts to behavior modification, caloric restriction, and exercise.

Between weeks 34 and 104, we explored different schema for administering fenfluramine plus phentermine in open-label fashion. At week 34, the original placebo group participants began taking fenfluramine plus phentermine (placebo-to-active group). Those receiving fenfluramine plus phentermine between weeks 6 and 34 either continued to receive medication or began targeted intermittent therapy. Participants who did not lose 10% of initial weight received an augmented dose (60 mg fenfluramine plus 30 mg phentermine. The placebo-to-active group lost an additional 9.1 +/- 0.8 kg (mean +/- SEM) in the period from week 34 to week 60. At week 60, they were assigned to either continue medication, intermittent therapy, or augmented therapy. More than 68% (83) of the original participants completed up to study week 104. At that point, overall weight loss was 10.8 +/- 0.7 kg (11.6 +/- 0.8% of initial weight); participants who continued to receive fenfluramine plus phentermine lost 11.6 +/- 0.8 kg, participants receiving intermittent therapy lost 11.6 +/- 1.3 kg, and participants receiving augmented therapy lost 6.5 +/- 1.5 kg. Although 41% of the participants complained of dry mouth, neither serious adverse effects nor evidence of medication abuse appeared. There were 29 dropouts in the period from weeks 34 to 104. Sixteen of those were related to medication (adverse effects, lack of efficacy, and fear of medication). Overall, fenfluramine plus phentermine used in conjunction with behavior modification, caloric restriction, and exercise continued to be efficacious for up to 2 years.

Long-term weight control study. III (weeks 104 to 156). An open-label study of dose adjustment of fenfluramine and phentermine.

Between weeks 104 and 156 we attempted to optimize response by adjusting the doses of fenfluramine and phentermine. Dosing changes were based on an algorithm that aimed to achieve 120% of ideal body weight (IBW) while minimizing adverse effects. The dose groups were as follows: stage I, 30 mg fenfluramine plus 15 mg phentermine in the morning; stage II--continuous or targeted intermittent, 60 mg fenfluramine plus 15 mg phentermine in the morning; stage III, 60 mg fenfluramine plus 30 mg phentermine in the morning; stage IV, 60 mg fenfluramine plus 30 mg phentermine in the morning and 30 mg fenfluramine in the evening; and stage V, 60 mg fenfluramine plus 30 mg phentermine in the morning and 60 mg fenfluramine in the evening. Seventy-seven participants began this segment of the study and 59 completed to week 156. Completers of this segment of the study gained an average of 2.7 +/- 0.5 kg between weeks 104 and 156 but remained 9.4 +/- 0.8 kg (10.5%) below baseline. On average, weight loss from baseline by group was as follows: for stage I (n = 2), 14.1 +/- 6.8 kg; for stage II continuous (n = 14), 10.9 +/- 0.7 kg; for stage II targeted intermittent (n = 7), 8.8 +/- 2.4 kg; for stage III (n = 9), 7.7 +/- 2.6 kg; for stage IV (n = 8), 10.5 +/- 2.6 kg; and for stage V (n = 19), 8.4 +/- 2.4 kg. Upward dose adjustment (n = 36) resulted in further weight loss in 11 and no gain in six participants.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term weight control study. IV (weeks 156 to 190). The second double-blind phase.

To assess continued efficacy of anorexiants after 3 years of use, 52 participants (43% of those starting) entered a second double-blind trial to compare 60 mg sustained-release fenfluramine plus 15 mg phentermine resin versus placebo added to behavior modification, caloric restriction, and exercise. Although participants in both the active medication and placebo groups gained weight, participants receiving fenfluramine plus phentermine (n = 27) gained significantly (p less than 0.01) less (4.4 +/- 0.5 kg or 5.3% +/- 0.5% of initial weight) than participants receiving placebo (n = 24) (6.9 +/- 0.8 kg or 8.5% +/- 1.1% of initial weight). At week 190, both groups were still below their initial weight (fenfluramine plus phentermine group, 5.0 +/- 1.4 kg; placebo group, 2.1 +/- 1.2 kg; p less than 0.01). Overall, 12 participants (23.5% of those still in the study) were greater than or equal to 10% below initial weight. One participant dropped out during this phase because of personal reasons and loss of medication efficacy. During the 30 weeks, participants receiving fenfluramine plus phentermine had 26 moderate or severe complaints versus eight participants receiving placebo. Fenfluramine plus phentermine provided better appetite control and only slightly more bother. Analysis of participant response in this phase by treatment assignment in the first double-blind phase (weeks 6 to 34) indicated that initial receipt of medication did not have negative learning effects. Eleven participants receiving active medication between weeks 6 and 34 and receiving placebo between weeks 160 to 190 gained 5.1 +/- 1.0 kg. In contrast, 13 participants originally taking placebo gained 8.3 +/- 9 kg in this second double-blind phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term weight control study. V (weeks 190 to 210). Follow-up of participants after cessation of medication.

Participants who completed up to week 190 in the long-term weight control study were monitored after cessation of medication between weeks 190 and 210. Caloric restriction, behavior modification sessions, exercise reinforcement, and physician visits continued. We assessed whether or not participants had reset their weight control mechanisms and compared the effect of stopping medication under open-label conditions (weeks 190 to 210) with the results of stopping anorexiants under double-blind conditions (weeks 160 to 190). At week 210, participants were, on average, 1.4 +/- 1.0 kg (mean +/- SEM, 1.5% +/- 1.1%) below their weights at baseline (week 0). Of the 48 participants who remained in the study, 13 were still 5% or more and seven were 10% or more below their initial weights. On average, participants gained 2.7 +/- 0.5 kg (3.2%) in the period from weeks 190 to 210. Those who had been taking medication in the period from weeks 160 to 190 gained weight at a somewhat faster rate than those who had been taking placebo. However, participants who had transferred from fenfluramine plus phentermine to no medication in this phase gained at a slower rate than participants who had changed from fenfluramine plus phentermine to placebo under double-blind conditions at week 160 (0.195 kg per week versus 0.277 kg per week). The findings indicate that participants had difficulty maintaining weight loss without anorexiant medications. Despite long periods of time at weights much lower than baseline, permanent resetting of weight control mechanisms could not be shown for most participants.

Long-term weight control study. VI. Individual participant response patterns.

We analyzed the individual response patterns of all 121 participants who entered the study. Fifty-one participants completed up to week 190. In 26 completers, the response pattern consisted of an initial beneficial effect (greater than or equal to 6 months of weight loss greater than or equal to 10% from baseline) and later success (weight loss of greater than or equal to 10% from baseline at week 160). These successful participants had lost 14.1 +/- 1.0 kg (mean +/- SEM); 15.9% +/- 0.9% of initial weight) at week 160 and 8.1 +/- 1.2 kg (9.1% +/- 1.3% of initial weight) at week 190. A second pattern observed in 16 completers consisted of initial benefit and later partial success (loss of 0.1% to 9.9% at week 160). Other response patterns observed in completers included showing initial benefit only (n = 3) and no success (n = 6). Seventy of the 121 participants left the study before week 190. There were 22 "dropout successes" who had consistent weight loss for 1 year or more and were greater than or equal to 10% below their initial weights at time of dropout. Fifteen "dropouts with initial benefit" stayed in the study for greater than or equal to 1 year with initial benefit (weight loss greater than or equal to 10% from baseline maintained for greater than or equal to 6 months). Medication-related reasons accounted for only 10 of the 37 dropouts in the group with initial or later benefit. Minimal benefit was seen in 17 dropouts. Another 16 were in the study less than 1 year. Analysis of individual participant responses made some other generalizations possible. Participants receiving continuous medication lost more weight and had fewer adverse effects than those receiving targeted intermittent medication. Upward dose adjustment appeared to help 24 participants achieve the criteria for late or partial success. Analyses of individual participant responses can suggest ways to optimize anorectic medication use for individual patients.

Long-term weight control study. VII (weeks 0 to 210). Serum lipid changes.

We analyzed serum total cholesterol, triglycerides, and lipoprotein profile changes occurring in the participants (N = 121) through 210 weeks of the study. On average, baseline lipid levels were within normal limits. The most consistent changes occurred in the high-density lipoprotein cholesterol (HDL-C), serum total cholesterol/HDL-C ratios, and triglyceride levels. HDL-C increased significantly (p less than 0.01), compared with baseline, by 10% at week 34, 15% at week 54, 19% at week 104, and 27% at week 139. At week 210, 20 weeks after treatment had ended, HDL-C was 15% higher than baseline. At weeks 34, 54, 104, and 139, the serum total cholesterol/HDL-C ratio was significantly decreased, compared with baseline, by 9%, 19%, 17%, and 25%, respectively. At week 210, serum total cholesterol/HDL-C ratio was 8% less than week 0. Compared with baseline, triglyceride levels decreased significantly by 21%, 31%, 29%, and 29% at weeks 34, 54, 104, and 139, respectively. At week 210, triglyceride levels were 16% below baseline. Total cholesterol levels and low-density lipoprotein cholesterol (LDL-C) showed less dramatic changes. Patterns of lipid and lipoprotein changes were qualitatively similar between men and women. However, greater decreases in serum total cholesterol, LDL-C, and triglyceride levels were observed in participants with high (n = 10) compared with low (n = 10) baseline lipid levels. Cholesterol changes were not affected by anorexiant medications. However, triglyceride levels at week 34 were significantly (p less than 0.025) less in the participants treated with anorexiants.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of vitamin A toxicity.

Toxicity has been associated with abuse of vitamin A supplements and with diets extremely high in preformed vitamin A. Consumption of 25,000-50,000 IU/d for periods of several months or more can produce multiple adverse effects. The lowest reported intakes causing toxicity have occurred in persons with liver function compromised by drugs, viral hepatitis, or protein-energy malnutrition. Certain drugs or other chemicals may markedly potentiate vitamin A toxicity in animals. Especially vulnerable groups include children, with adverse effects occurring with intakes as low as 1,500 IU.kg-1.d-1, and pregnant women, with birth defects being associated with maternal intakes as low as approximately 25,000 IU/d. The maternal dose threshold for birth defects cannot be identified from present data. An identifiable fraction of the population surveyed consumes vitamin A supplements at 25,000 IU/d and a few individuals consume much more. beta-Carotene is much less toxic than vitamin A.

Reserpine up-regulation of rat renal cortical beta adrenergic receptors is independent of its effect on the sympathetic nervous system.

Experiments were performed to evaluate the mechanism underlying our recent observation that reserpine but not surgical denervation up-regulates rat renal cortical beta adrenergic receptors. The specific binding of [125I]iodocyanopindolol was used to quantitate the beta adrenoceptors. Chronic high-dose guanethidine, which decreased renal tissue and circulating catecholamines to the same extent as reserpine, failed to up-regulate renal beta adrenoceptors, which indicates that the effect of reserpine was not due to changes in the ambient catecholamine concentration. Isoproterenol-stimulated renin secretion, a measure of postsynaptic receptor function, was increased by reserpine but not denervation, which indicates that the failure to observe beta adrenoceptor up-regulation by radioligand binding studies after denervation was not an anomaly caused by loss of presynaptic receptors masking postsynaptic supersensitivity. Reserpine was effective even in denervated kidneys. Up-regulation of renal beta adrenoceptors with reserpine occurred even after destruction of peripheral sympathetic nervous system by a combination of adrenal demedullation and high-dose guanethidine administration. A lower daily dose of reserpine (0.3 mg/kg instead of 0.5 mg/kg), which caused no weight loss, was effective in producing beta adrenoceptor up-regulation. Antagonism of reserpine depletion of nerve terminal norepinephrine by tranylcypromine, a monoamine oxidase inhibitor, did not nullify renal beta adrenoceptor up-regulation. Overall, our results indicate that reserpine up-regulation of renal beta adrenergic receptors is independent of the sympathetic nervous system and possibly is a direct effect.

Reserpine but not surgical denervation regulates rat renal beta-adrenergic receptors.

The effects of unilateral surgical denervation or reserpine administration on renal beta-adrenergic receptors were examined in rat kidney cortex. The specific binding of [125I]iodocyanopindolol was used to quantitate the beta-adrenoceptors. Denervation had no significant effect on beta-adrenoceptor concentration in denervated compared with contralateral control kidney, 7 days postsurgery. In contrast, reserpine treatment increased beta-adrenoceptor concentration 30% compared with control (P less than 0.05). Tissue norepinephrine levels were depleted to a significant extent with both manipulations. The reserpine effect was investigated further. Reserpine increased both beta 1- and beta 2-adrenergic receptor subtypes to the same extent. The effect of reserpine was primarily on tubular beta-adrenoceptors including those in the proximal tubules; glomerular beta-adrenoceptors were minimally affected by reserpine. Other adrenergic receptor subtypes (alpha 1- and alpha 2-) were also significantly increased by reserpine; however, angiotensin II receptors were not altered, indicating that the reserpine effect was not a general one affecting all membrane receptors. Reserpine treatment increased beta-adrenergic receptor-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by 49% over control in the renal cortex. Denervation had no significant effect on cAMP accumulation. Overall, our results suggest that, in addition to sympathetic nerve terminal norepinephrine, other factors may be involved in the regulation of renal beta-adrenergic receptors.

Sympathetic neural modulation of the immune system. I. Depression of T cell immunity in vivo and vitro following chemical sympathectomy.

Chemical sympathectomy of adult mice with 6-hydroxydopamine (6-OHDA) either prior to or following epicutaneous sensitization with the trinitrophenyl (TNP) hapten decreased the delayed hypersensitivity (DH) response to ear challenge. To determine if uptake of 6-OHDA into sympathetic nerve terminals, and their subsequent destruction, was required for suppression of DH, the catecholamine uptake blocker, desipramine, was employed to block 6-OHDA-induced sympathetic denervation. Pretreatment with desipramine prevented the depression of DH. In vivo treatment with the beta blocker, propranolol, did not alter the 6-OHDA effect, eliminating the potential contribution of released catecholamines, acting on beta-adrenoceptors, to DH reduction. Sympathectomy before sensitization also diminished hapten-specific T cell reactivity of sensitized lymph node (LN) cells, as measured in vitro by IL-2 production and CTL generation. In vivo DNA synthesis in draining LN in response to immunization was modestly decreased following 6-OHDA. Thus, sympathetic denervation appears to impair T cell activity in vivo and in vitro. Overall, these results indicate the SNS plays a role in generation of cell-mediated immunity.

Glomerular and tubular alpha 1- and alpha 2-adrenoceptors in the rat kidney: distribution in basolateral and brush border membranes of tubular cells.

Rat kidney glomeruli and cortical tubules were obtained by a combination of sieving and differential centrifugation technique. [3H]Prazosin and [3H]rauwolscine were used to identify and quantify the alpha 1- and alpha 2-adrenergic receptors, respectively. In the glomeruli, the alpha 1-adrenoceptor concentration was 27% and alpha 2-adrenoceptor concentration was 33% of the corresponding values in the tubules. Further localization of the tubular alpha-adrenoceptors was undertaken by studies in the isolated basolateral membrane and comparison with values in the crude plasma membrane. alpha 1-Adrenoceptors were enriched 1.54 +/- 0.1 times and alpha 2-adrenoceptors were enriched 1.73 +/- 0.04 times in the basolateral membrane as compared to crude plasma membrane. However, these values were significantly (p less than 0.05) less than the enrichment value of 2.77 +/- 0.3 obtained for the basolateral membrane marker (Na+ + K+)-ATPase. These results suggested the possibility that alpha 1- and alpha 2-adrenoceptors may also be distributed in the brush border membrane. Direct-binding studies in the purified renal brush border membrane indicated alpha 1-adrenoceptor concentration of 82.1 +/- 3.8 and alpha 2-adrenoceptor concentration of 108.2 +/- 9.3 fmol/mg protein. These values were 32 and 17% of the corresponding values in the basolateral membrane. Overall, our results suggest that alpha 1- and alpha 2-adrenoceptors are present both in the basolateral and brush border membranes analogous to what has been reported for angiotensin and insulin receptors; their primary concentration, however, is in the basolateral membranes.

Adrenergic receptors on cerebral microvessels: pericyte contribution.

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

Beta-adrenergic receptor coupled adenylate cyclase in rat kidney. Differential coupling in glomeruli and tubules.

[125I]Iodocyanopindolol ([125I]ICYP) was used to identify and characterize the beta-adrenoceptors in isolated rat kidney glomeruli and cortical tubules. In both the tissues, specific binding of [125I]ICYP was saturable with time and ligand concentration and showed appropriate stereospecificity and agonist rank order potency characteristic of binding to beta-adrenoceptors. Scatchard analysis revealed that the beta-adrenoceptor concentration in the glomeruli (111.1 +/- 8.9 fmol/mg protein) was about three times that in the tubules (40.1 +/- 1.8 fmol/mg protein). The dissociation constants (KD) were similar in the two tissues. Both beta 1- and beta 2-adrenoceptor subtypes were present in the glomeruli and tubules, but the beta 1-subtype was predominant, constituting greater than 80% of the total beta-adrenoceptors in the two tissues. Isoproterenol was twice as potent in competing for [125I]ICYP binding in the tubules as in the glomeruli (P less than 0.05). The slope factor (pseudo-Hill coefficient) for the isoproterenol competition curve was 0.74 +/- 0.04 in the glomeruli and 0.54 +/- 0.02 in the tubules (P less than 0.05). The nonmetabolized guanyl nucleotide analogue Gpp(NH)p caused a steepening and a 3-fold shift of the isoproterenol competition curve in both tissues. Isoproterenol-stimulated cAMP accumulation in the glomeruli was only 31% of the value in the tubules. The concentration of isoproterenol producing half-maximal stimulation (EC50) was 114 +/- 13 nM in the glomeruli and 19 +/- 3 nM in the tubules (P less than 0.05). Gpp(NH)p and forskolin caused a similar increase in cAMP accumulation over basal value in the glomeruli as in the tubules. Overall, our results indicate a decreased efficiency in the interaction between the beta-adrenergic agonist hormone, the receptor and the guanine nucleotide regulatory protein in the glomeruli as compared to the tubules.

Cellular retinyl esters and retinol among parenchymal and stellate cells in normal rat liver.

[11,12-3H] Retinyl acetate (100 micrograms/20 microCi/rat) in corn oil was fed by stomach tube to normal male Wistar-Furth rats (approximately 250 g body weight). After 15 days, the contents of retinyl esters and retinol (total retinol) and their 3H-radioactivity were measured in the whole liver, crude parenchymal cells and the purified parenchymal cells, employing differential centrifugation, centrifugal elutriation and high performance liquid chromatography (HPLC) techniques. Of the total liver retinol (nmol/g liver), the crude parenchymal cells had nearly 90%, whereas the purified parenchymal cells had only 21% based on HPLC analysis. Furthermore, of the total liver retinol radioactivity (dpm/g liver) the crude parenchymal cell fraction had 85%, while the purified parenchymal cell fraction had only 16%. Based on the cell number, the crude parenchymal cell fraction was contaminated by retinoid-rich stellate cells to the extent of 4%. It, therefore, was concluded that the parenchymal cells accounted for 16-21%, whereas the stellate cells contributed 79-84% of total retinol stored in the liver under normal steady-state conditions. It also was calculated that on a per mg basis, stellate cells had 200 times more total retinol than parenchymal cells, whereas on a per cell basis each stellate cell had 74 times more total retinol than a parenchymal cell.