Dihydroxyphenylglycol and intraneuronal metabolism of endogenous and exogenous norepinephrine in the rat vas deferens

To elucidate the origin and significance of dihydroxyphenylglycol (DHPG) as a metabolite of norepinephrine (NE), the isolated rat vas deferens was preloaded with tracer amounts of tritiated NE and examined for the release of radioactive and endogenous NE and DHPG before and during electrical stimulation or stimulation with excess K+. Tissues were incubated with desipramine or reserpine to determine the effects of blockade of neuronal uptake and of interference with vesicular translocation of NE. Radioactive NE appeared to distribute differently from endogenous NE into at least two pools, but for the most part endogenous NE and DHPG behaved similarly in response to pharmacological manipulations. Desipramine blocked completely the increased appearance of both radioactive and endogenous DHPG in the medium during electrical stimulation or K+ stimulation; DHPG responses to stimulation are thus dependent on recapture of NE at the synapse. Basal release of DHPG was increased by reserpine, and this increase was not affected by desipramine; therefore, reserpine-induced release of DHPG is independent of neuronal uptake consistent with formation of DHPG from NE leaking into the cytosol from vesicular stores. Reserpine enhanced the release of DHPG during stimulation, and concomitant desipramine treatment blocked this effect; thus, interference with NE translocation into storage vesicles increases the availability of recaptured NE for intraneuronal metabolism. During stimulation of NE release between 70 to 80% of the recaptured NE was estimated to be sequestered into storage vesicles for rerelease. Combined measurement of endogenous and labeled NE and DHPG provides a useful tool for examining neuronal uptake and intraneuronal disposition of NE.     

Urinary excretion of dihydroxyphenylalanine and dopamine during alterations of dietary salt intake in humans

1. Urinary excretion of dopamine (DA) increases during dietary salt loading. The majority of urinary DA is derived from circulating dihydroxyphenylalanine (dopa). Whether the increase in urinary DA excretion during salt loading results from increased efficiency of uptake of dopa by proximal tubular cells of the kidney, facilitation of intracellular conversion of dopa to DA, or increased delivery of dopa to tubular uptake sites, has been unknown. 2. In 10 inpatient normal volunteers on a constant diet, daily excretion of dopa and DA was assessed during normal sodium intake (109 mmol/day) for 1 week, low sodium intake (9 mmol/day) for 1 week and high sodium intake (249 mmol/day) for 1 week. 3. Urinary DA excretion exceeded urinary dopa excretion by about tenfold, and the excretion of both DA and dopa increased by about twofold between the low and high salt diets, with similar proportionate changes. Plasma dopa was unchanged by dietary salt manipulation. 4. The results indicate that increases in urinary DA excretion during dietary salt loading can be accounted for by increased delivery of dopa to sites of uptake by proximal tubular cells. Since dopa is released into the bloodstream by sympathetic nerve endings and by the brain, and since interference with decarboxylation of dopa attenuates natriuretic responses, dopa may function indirectly as a neurohormone involved in homoeostatic regulation of sodium balance.     

Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites

Acute beta-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P<0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD22,23 (P<0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.     

Polymorphism of adhesion molecule CD31 is not a significant risk factor for graft-versus-host disease

Mismatch between bone marrow transplant (BMT) patient and donor for an amino acid polymorphism within the adhesion molecule CD31 has recently been reported to increase risk for the development of graft-versus-host disease (GVHD). We further examined this association in a larger series of 301 BMT patients (227 with grade III/IV GVHD and 74 with grade 0 GVHD) and their HLA-identical sibling donors. CD31 genotypes were determined by polymerase chain reaction and restriction endonuclease digestion. The role of mismatch at the CD31 locus in the development of GVHD was assessed by analyzing the extent of CD31 identity and CD31 compatibility among the grade 0 GVHD and grade III/IV GVHD sibling pairs. No significant association between CD31 mismatch and the development of severe GVHD was detected in our overall patient population. Sixty-three percent of grade III/IV GVHD sibling pairs and 69% of grade 0 GVHD sibling pairs had CD31 genotypes that were identical (P = .36, odds ratio = 1.30). In addition, neither the grade 0 GVHD group (P = .10) nor the grade III/IV GVHD group (P = .27) differed significantly from the expected probability of identity between sibling pairs. Mismatch at the CD31 polymorphism between recipients and donors showed no consistent association with the development of GVHD. Current evidence does not support the value of CD31 mismatch in the selection of BMT donors.     

Inhibition of delayed-type contact hypersensitivity in mice deficient in both E-selectin and P-selectin

Leukocyte rolling and emigration in response to inflammatory stimuli appears to involve both E-selectin- and P-selectin-dependent adhesion, which suggests that these molecules have overlapping functions. To clarify their relative contributions in chronic inflammation, we examined delayed-type contact hypersensitivity (DTH) responses in P- selectin, E-selectin, and E-/P-selectin-deficient mice. Oxazolone- induced increases in ear thickness and ear weight were equivalent in wild-type mice and in P-selectin and E-selectin mutants, but were significantly reduced in E-/P-selectin mutants. The number and area of microabscesses on the ears of E-/P-deficient mice were decreased by 72% and 93%, and the number of leukocytes invading the subdermal ear tissue was reduced. T cells from E-/P-deficient mice transferred oxazolone reactivity into naive wild-type mice. However, when donor T cells from wild-type mice were transferred into E-/P-selectin-deficient mice, the DTH response was significantly impaired. These results show that leukocyte recruitment into a subacute inflammatory reaction can occur when either P-selectin or E-selectin is present, but is significantly reduced when both selectins are absent. Both P- and E-selectin are likely to play important roles in the development and maintenance of inflammatory diseases.     

Impaired metabolic modulation of α-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate α-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of α-adrenergic vasoconstriction in active skeletal muscle.     

Effects of nafazatrom on cardiovascular, sympathetic, and endocrine responses to hemorrhagic shock in conscious rats

Nafazatrom is an antithrombic drug that has been shown to have beneficial effects in traumatic shock and organ ischemia. This study evaluated the effect of nafazatrom on cardiovascular, sympathetic, and endocrine consequences to moderate or severe hemorrhagic shock in the conscious rat. Nafazatrom (2 mg/kg, i.v.) had no effect on the blood pressure, heart rate, and circulatory norepinephrine, vasopressin, and leukotriene C4 responses to bleeding. Nafazatrom significantly reduced plasma TXB2 and 6-keto-PGF1 alpha and blocked the increment in these cyclooxygenase metabolites in response to hemorrhage. It is concluded that nafazatrom does not increase survival after moderate hypovolemic hypotension and decreases survival to severe hemorrhage. Nafazatrom does not modify the cardiovascular, sympathetic, and neuroendocrine responses to hypovolemic hypotension.     

Neuronal source of plasma dihydroxyphenylalanine

The source and significance of plasma levels of dihydroxyphenylalanine (DOPA), the precursor of the endogenous catecholamines, have been unknown. We measured arterial and venous plasma DOPA concentrations in healthy subjects at rest, patients who had undergone regional sympathectomies or were undergoing general anesthesia, and subjects during procedures (tilt, oral clonidine, or iv isoproterenol, yohimbine, trimethaphan, or diazepam) known to affect plasma norepinephrine levels. We also measured plasma DOPA in laboratory animals during anesthesia, after adrenalectomy, or after administration of alpha-methyl-para-tyrosine, which competitively inhibits tyrosine hydroxylase, the intraneuronal enzyme catalyzing the rate-limiting step in catecholamine biosynthesis. In virtually all healthy subjects there was an arteriovenous increment in plasma DOPA (mean increase, 32%; P less than 0.001), whereas in sympathectomized patients there was not (mean decrease, 16%; P less than 0.001 compared with healthy subjects). Except for small decreases after clonidine treatment, none of the above procedures affected plasma DOPA levels. Plasma DOPA decreased during general anesthesia and returned to baseline upon reversal of the anesthesia. Adrenalectomy had no effect on plasma DOPA. alpha-Methyl-para-tyrosine decreased plasma DOPA by 62% (P less than 0.01). The results support the suggestion that DOPA can pass across sympathetic neuronal membranes to reach the general circulation. If so, then the regional rate of appearance of DOPA in plasma may be related to the regional rate of tyrosine hydroxylation. Conversely, DOPA taken up from the circulation may provide a source for catecholamine biosynthesis in tissues devoid of tyrosine hydroxylase.     

Relationships between urinary inositol excretions and whole-body glucose tolerance and skeletal muscle insulin receptor phosphorylation

This study assessed the relationships of urinary d-chiro-inositol and myo-inositol excretions to indices of whole-body glucose tolerance and total content and tyrosine phosphorylation of the insulin receptor (activation) in skeletal muscle of older nondiabetic subjects. Fifteen adults (age, 65 ± 8 years; body mass index, 27.9 ± 3.3 kg/m² [mean ± SD]) completed duplicate assessments of oral (75-g oral glucose tolerance test [OGTT]) and intravenous (300 mg/kg body weight intravenous glucose tolerance test) glucose tolerance challenges and 24-hour urinary d-chiro-inositol and myo-inositol excretions. Skeletal muscle (vastus lateralis) biopsies were obtained at minute 60 of the OGTTs. Subjects with higher urinary d-chiro-inositol excretion had higher insulin (ρ = 0.51, P ≤ .05) and C-peptide (ρ = 0.56, P ≤ .05) area under the curves, and lower insulin sensitivity index (ρ = −0.60, P ≤ .05) during the intravenous glucose tolerance test. The urinary myo- to d-chiro-inositol ratio was also inversely related to insulin area under the curve (ρ = −0.59, P ≤ .05). Urinary d-chiro-inositol (ρ = −0.60, P ≤ .05) and myo-inositol (ρ = −0.60, P ≤ .05) were inversely related to tyrosine phosphorylation of the insulin receptor (phosphotyrosine 1162/1163), but not total content of the insulin receptor during the OGTT. The apparent relationships were modestly weakened when adjustments were made for sex. These findings support previous research linking higher urinary d-chiro-inositol excretion with a progressive decline in whole-body glucose tolerance. This is the first report to link higher urinary d-chiro-inositol excretion to a blunted activation of skeletal muscle insulin receptor signaling in older nondiabetic subjects.