Hyperinnervation of mesenteric arteries in spontaneously hypertensive rats by sympathetic but not primary afferent axons

Hypertrophy of the perivascular plexus is thought to play a role in the development of hypertension in spontaneously hypertensive rats (SHR). However, it is not known whether the sympathetic varicosities are more numerous or larger, or form more neurovascular junctions. Further, a parallel hypertrophy of primary afferent terminals around the vessels might modulate any effects of hypertrophied sympathetic terminals. We have investigated the perivascular plexus around second-order mesenteric arteries of SHR and Wistar-Kyoto (WKY) rats by electron microscopy. Noradrenergic terminals were identified by the presence of small granular vesicles after chromaffin fixation, and substance P (SP+) afferent axons were identified by immunohistochemistry. The numbers of noradrenergic axon and varicosity profiles were higher (48 and 25%, respectively) in SHR than in WKY rats, and the majority lay closer to the medio-adventitial border. In contrast, there was no difference in the numbers of SP+ axons. Sympathetic and SP+ varicosities were indistinguishable in size, shape, vesicle content and mitochondrion content between each other and between the strains. However, both the number of neuromuscular junctions and the proportion of varicosities that formed them in SHR arteries were more than double those in WKY vessels. The data clearly show that hyperinnervation in SHR is specific for noradrenergic axons.     

Endothelin-1 potentiates cholinergic nerve-mediated contraction in human isolated bronchus.

That endothelin-1(ET-1) plays a mediator role in asthma is consistent with reports of ET-1-induced potentiation of cholinergic nerve-mediated contraction in airways from various animal species. This study examined the effect of ET-1 on cholinergic contractions in human isolated bronchus. Macroscopically nondiseased human bronchial tissue was obtained from 23 patients with respiratory tumours. An electrical field stimulation (EFS) frequency that produced one third of the contraction at 30 Hz (EFS30) was estimated. The effect of ET-1 on these EFS-evoked contractions was assessed. EFS-evoked contractions were frequency-dependent and abolished by either atropine or tetrodotoxin. Thus, EFS-induced contractions were mediated by acetylcholine from cholinergic nerves. ET-1 (3 nM) potentiated EFS-evoked contractions by 10+/-2% EFS30 (p<0.05) without any significant effect on contractions induced by exogenous acetylcholine. Neither the ET(A) receptor-selective antagonist BQ-123 (3 microM) nor the ET(B) receptor-selective antagonist BQ-788 (10 microM) alone significantly altered ET-1-induced potentiation of EFS-evoked contractions. However, in the combined presence of both BQ-123 and BQ-788, ET-1-induced potentiation of EFS-evoked contractions was abolished. Thus, prejunctional endothelinA and endothelinB receptors appear to mediate endothelin-1-induced potentiation of electrical field stimulation-evoked cholinergic contractions in human bronchus. This suggests another potentially important mechanism through which endothelin-1 could increase bronchial tone in asthma.     

Some implications of the high intrasynaptic norepinephrine concentrations in resistance arteries

Norepinephrine concentrations in excess of 10(-4) M are achieved at the post-junctional membrane of the adrenergic synaptic cleft in resistance arteries at least for short periods of time. These concentrations are greater than the threshold of low affinity non-alpha-adrenoceptors-'extraceptors', that respond to norepinephrine and would be sufficient to overcome the blockade by alpha-adrenoceptor antagonists. The relationship of such activity to the excitatory junction potential and the relevance of this to contraction in vivo remains a matter of conjecture.     

Preserved norepinephrine reuptake but reduced sympathetic nerve endings in hypertrophic volume-overloaded rat hearts

BackgroundIn congestive heart failure (CHF), an activation of the cardiac sympathetic nervous system results in depleted cardiac norepinephrine (NE) stores. The underlying regulatory mechanisms are discussed controversially and were investigated in the present study in CHF resulting from volume overload.Methods and ResultsAorto-caval shunt (AVS) was performed in rats. Plasma NE levels were determined by radioenzymatic assay, left ventricular NE by high-performance liquid chromatography, endothelin-1 by enzyme-linked immunosorbent assay. Tyrosine-hydroxylase (TH)– and nerve growth factor (NGF)–mRNA was determined by Northern blot analysis and ribonuclease-assay. Cardiac [³H]-NE uptake was measured in isolated perfused hearts. Glyoxylic acid–induced histofluorescence was used to quantify cardiac sympathetic nerves. Compared with sham-operated animals (SH), AVS rats were characterized by depleted cardiac NE stores and enhanced NE plasma levels. Neither TH-mRNA levels in stellate ganglia, nor cardiac [³H]-NE-uptake were reduced in AVS. The left ventricular density of sympathetic nerves was markedly decreased. Gene expression of myocardial NGF (a positive regulator of NE reuptake and cardiac sympathetic nerve density) and left ventricular endothelin-1 (a negative regulator of NE reuptake and positive regulator of cardiac NGF expression) were unchanged.ConclusionIn volume-overloaded hypertrophic hearts, depletion of cardiac NE stores is caused by a reduction of the sympathetic nerve density, whereas cardiac NE reuptake is preserved.     

Inhibitors of actin filament polymerisation attenuate force but not global intracellular calcium in isolated pressurised resistance arteries

Receptor-coupled contractile activation of arterial smooth muscle involves increases in intracellular calcium ([Ca(2+)](i)) and subsequent alteration of myosin light chain phosphorylation. An additional mechanism whereby agonists could regulate vascular contractility may be alteration of actin filament dynamics. Therefore, in this study, we have investigated the influence of two inhibitors of actin filament polymerisation, cytochalasin D and latrunculin B, on the [Ca(2+)](i) and force responsiveness of pressurised rat mesenteric arteries to alpha-adrenergic stimulation. Following cytochalasin D or latrunculin B treatment, phenylephrine-induced constrictions were significantly reduced to 11 +/- 3.2% (n = 6) and 10 +/- 4.4% (n = 6) of control, respectively, whereas [Ca(2+)](i) remained at 98 +/- 21% and 104 +/- 7.0% of control, respectively. Such effects of cytochalasin D were not restricted to mesenteric small arteries. Cytochalasin D also significantly reduced the force, but not [Ca(2+)](i) responses to agonist stimulation in other vascular (portal vein) and non-vascular (uterine) tissues. These data indicate that inhibitors of net actin polymerisation attenuate maximum agonist-induced force responsiveness without similar reductions in [Ca(2+)](i) in pressurised resistance vessels and other smooth muscle tissues. This suggests that modulation of the dynamic equilibrium between filamentous F-actin and monomeric globular actin (G-actin) may be an important mechanism, acting independently of global [Ca(2+)](i) homeostasis, to regulate the smooth muscle contractile state.     

Prejunctional and postjunctional actions of endogenous norepinephrine at the sympathetic neuroeffector junction in canine coronary arteries

The effects of endogenous and of exogenous norepinephrine were studied in isolated rings of canine left circumflex coronary artery and its first ventricular branch. Norepinephrine was released from adrenergic nerve endings by transmural electrical stimulation and by tyramine. In rings contracted with prostaglandin F2 alpha, transmural electrical stimulation resulted in frequency-dependent relaxations which were blocked by propranolol or tetrodotoxin; tyramine and exogenous norepinephrine caused concentration-dependent relaxations which were blocked by propranolol. The tyramine-induced relaxations also were inhibited by cocaine. The left circumflex artery was less sensitive than its branch to beta-adrenergic activation; this difference was significant even between rings of the two vessels immediately adjacent to the branching point and was abolished by phentolamine. In the presence of propranolol, transmural electrical stimulation, tyramine and phenylephrine, produced contractions of the left circumflex artery, but not the branch; these contractions were prevented by phentolamine. Phentolamine, but not prazosin, augmented the beta-adrenergic response of left circumflex artery to low frequency stimulation; in arteries preincubated with 3H-norepinephrine, this was accompanied by an increased overflow of tritiated neurotransmitter. The prejunctional effect of phentolamine was also evident in branch coronary arteries which exhibit no postjunctional alpha-adrenergic responses. With high frequency stimulation, both alpha-adrenergic antagonists equally augmented the relaxation of left circumflex artery; the efflux of tritiated norepinephrine was not different from untreated arteries. These experiments demonstrate, in isolated coronary arteries, that the primary adrenergic response to released endogenous norepinephrine is beta-adrenergic relaxation. The prejunctional effects of nonspecific alpha-adrenergic antagonists preclude their use in determining the importance of postjunctional coronary alpha-adrenergic receptor activation caused by sympathetic nerve stimulation.     

O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells

OBJECTIVE: Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen. METHODS: Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. RESULTS: Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells. CONCLUSION: These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.     

Brainstem norepinephrine neurons mediate ethanol-evoked pressor response but not baroreflex dysfunction

BACKGROUND: Ethanol elicits strain-dependent blood pressure and baroreflex sensitivity responses in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats; the mechanisms underlying these divergent effects are not clear. The authors tested the hypothesis that differential neuronal actions of ethanol may account for these strain-dependent responses. To this end, the authors investigated the direct effects of ethanol on norepinephrine (NE)-containing neurons in the rostral ventrolateral medulla (RVLM), which modulate sympathetic neuronal activity, and on c-Jun-expressing neurons in the nucleus tractus solitarius (NTS), whose activity is inversely correlated with baroreflex sensitivity. METHODS: In a newly developed model system in conscious, freely moving rats, the effect of intra-RVLM or intra-NTS ethanol was investigated on neuronal NE at the microinjection site (in vivo electrochemistry), blood pressure, heart rate, spontaneous baroreflex sensitivity, and c-Jun expression in the NTS. RESULTS: Ethanol (1, 5, or 10 microg) microinjection into the RVLM elicited dose-dependent increases in RVLM NE and blood pressure in SHRs but not in WKY rats. Ethanol had no effect on the activity of the NE-containing neurons in the NTS of either strain. However, baroreflex dysfunction elicited by intra-NTS ethanol in conscious WKY rats was associated with enhanced expression of c-Jun in the NTS. CONCLUSIONS: (1) Ethanol activation of the NE-containing neurons in the RVLM of SHRs contributes to the centrally mediated pressor response, (2) the NE-containing neurons in the NTS are not involved in ethanol-induced baroreflex dysfunction, and (3) direct activation of the c-Jun-containing neurons in the NTS is implicated in baroreflex dysfunction elicited by ethanol in normotensive rats.     

Yohimbine increases cerebrospinal fluid and plasma norepinephrine but not arginine vasopressin in humans

Previous studies have demonstrated alpha 2-inhibitory regulation of central nervous system (CNS) noradrenergic and arginine vasopressinergic systems. We tested the hypothesis that alpha 2-inhibition of CNS noradrenergic and vasopressinergic systems is tonic in nature by measuring the response of cerebrospinal fluid (CSF) norepinephrine (NE) and arginine vasopressin (AVP) to the alpha 2-antagonist yohimbine in 7 young normal male human subjects. We also evaluated the tonic nature of alpha 2-inhibition of the sympathetic nervous system (SNS) and of AVP release into plasma by measuring the response of plasma NE and plasma AVP to yohimbine. CSF NE was significantly higher following yohimbine as compared to placebo. In contrast CSF AVP did not differ between yohimbine and placebo conditions. Similarly, plasma NE was significantly higher following yohimbine as compared to placebo, while plasma AVP was unchanged. These results support a tonic alpha 2-inhibitory regulatory mechanism for both CNS noradrenergic systems and sympathetic outflow. Such tonic alpha 2-inhibition could not be demonstrated for regulation of AVP levels in CSF or plasma in humans.     

Exercise training lowers resting renal but not cardiac sympathetic activity in humans

Endurance exercise training has previously been shown to reduce the plasma concentration of norepinephrine. Whether reduction in sympathetic activity is responsible for the blood pressure-lowering effects of exercise training is unknown. Using a radiotracer technique, we measured resting total, cardiac, and renal norepinephrine spillover to plasma in eight habitually sedentary healthy normotensive men (aged 36 +/- 3 years, mean +/- SEM) after 1 month of regular exercise and 1 month of sedentary activity, performed in a randomized order. One month of bicycle exercise 3 times/wk (40 minutes at 60-70% maximum work capacity) reduced resting blood pressure by 8/5 mm Hg (p less than 0.01) and increased maximum oxygen consumption by 15% (p less than 0.05). The fall in blood pressure was attributable to a 12.1% increase in total peripheral conductance. Total norepinephrine spillover to plasma was reduced by 24% from a mean of 438.8 ng/min (p less than 0.05). Renal norepinephrine spillover fell by an average of 41% from 169.4 ng/min with bicycle training (p less than 0.05), accounting for the majority (66%) of the fall in total norepinephrine spillover. Renal vascular conductance was increased by 10% (p less than 0.05), but this constituted only 18% of the increase in total peripheral conductance. There was no change in cardiac norepinephrine spillover. The reduction in resting sympathetic activity with regular endurance exercise is largely confined to the kidney. The magnitude of the fall in renal vascular resistance, however, is insufficient to directly account for the blood pressure-lowering effect of exercise, although other effects of inhibition of the renal sympathetic outflow may be important.     

Pyridostigmine potentiates L-dopa- but not arginine- and galanin-induced growth hormone secretion in children.

The coadministration of growth hormone (GH) secretagogues can provide insight into the neuroregulation of GH secretion. The GH response to L-dopa (125, 250 and 500 mg orally for body weights less than 15 kg, between 15 and 30 kg and greater than 30 kg, respectively), arginine (Arg; 0.5 g/kg infused intravenously over 30 min) and galanin (GAL; 15 micrograms/kg infused intravenously over 60 min) when administered alone or combined with pyridostigmine (PD; 60 mg orally), a cholinergic agonist that likely acts via inhibition of endogenous somatostatin secretion, was studied in children with familial short stature. The GH-releasing effect of PD was also evaluated. In 8 children, PD and L-dopa when administered alone induced an equivalent GH rise (area under the response curve, mean +/- SEM: 241.4 +/- 31.1 vs. 202.9 +/- 38.6 micrograms/l/h) while their coadministration had an additive effect (435.4 +/- 41.4 micrograms/l/h; p less than 0.02 vs. PD and L-dopa alone). On the contrary, in other 8 children, PD and Arg induced similar GH increases either when administered alone (394.2 +/- 68.5 vs. 405.8 +/- 103.9 micrograms/l/h) or in combination (535.8 +/- 97.3 micrograms/l/h). GH increases almost superimposable were also observed when PD and GAL were administered alone (405.2 +/- 72.3 vs. 412.6 +/- 94.1 micrograms/l/h) or in combination (537.9 +/- 139.0 micrograms/l/h) in other 7 children. These data show that the enhancement of the cholinergic activity by PD increases the L-dopa-induced GH release but fails to modify both Arg- and GAL-induced GH release in short children.(ABSTRACT TRUNCATED AT 250 WORDS)     

Piglet pial arteries respond to N-methyl-D-aspartate in vivo but not in vitro

Controversy exists concerning whether activation of N-methyl-D-aspartate (NMDA) receptors exerts direct dilator effects on cerebral arteries. The purpose of this study was to examine the responses of isolated piglet arteries to NMDA to determine whether isolated arteries, apart from surrounding neuronal tissue, are capable of responding to NMDA. Piglet arteries (100-200 microm) were isolated from branches of the middle cerebral artery and carefully dissected free of adherent tissue. Arteries were then mounted in an arteriograph system and pressurized to either 30 mm Hg (n=8), 60 mm Hg (n=10), 80 mm Hg (n=6), or 100 mm Hg (n=5). After development of spontaneous tone, NMDA (10(-5) to 10(-3) M) was administered abluminally to the vessels, and no appreciable response was noted (for example; 10(-4) M, 30 mm Hg: 3+/-3% change in active diameter; 60 mm Hg: -4+/-3% change in active diameter). Following a thorough washout, vessels were treated with bradykinin (10(-9) to 10(-7) M), and the arteries did respond (10(-7) M, 30 mm Hg: 26+/-3% change in active diameter; 60 mm Hg: 65+/-10% change in active diameter). In contrast, 10(-5) M and 10(-4) M NMDA dilated arteries in vivo by 9+/-2% and 29+/-6% change in active diameter, respectively (n=6). These results demonstrate that isolated cerebral arteries do not respond directly to NMDA receptor activation. This work confirms our previous in vivo data and is consistent with the hypothesis that cerebral arteries respond to NMDA through a secondary interaction mediated by neuronal release of NO and not to NMDA directly.     

Insulin potentiates TRPC3-mediated cation currents in normal but not in insulin-resistant mouse cardiomyocytes

OBJECTIVE: Recent studies show that bioactive lipids alter intracellular Ca(2+) handling of cardiac cells differently in normal and insulin-resistant cardiomyocytes. In the present study we measured non-selective cation currents (NSCC) focusing on the interaction between insulin, the bioactive lipid diacylglycerol (DAG) and canonical transient receptor potential 3 (TRPC3) channels. METHODS: Whole cell patch-clamp was used to measure NSCC in ventricular cardiomyocytes isolated from adult wild-type (WT) and insulin resistant, obese ob/ob mice. Western blot, immunoprecipitation and immunofluorescence staining were used to study the concentration and cellular distribution of TRPC3 channels. RESULTS: Application of the membrane permeable DAG analogue (OAG, 30 microM) induced an NSCC, which was approximately 40% smaller in ob/ob than in WT cardiomyocytes. Insulin induced a small NSCC with similar amplitude in ob/ob and WT cells. Pretreatment with insulin (60 nM) increased the OAG-induced NSCC in WT (by approximately 50%) but not in ob/ob cells. OAG-induced currents were inhibited by adding anti-TRPC3 antibodies to the patch pipette solution. The expression of TRPC3 was lower in ob/ob than in WT cardiomyocytes. TRPC3 was detected in glucose transporter 4 (GLUT4) immunoprecipitates. Insulin exposure resulted in a translocation of TRPC3 to the plasma membrane in WT but not in ob/ob cardiomyocytes. CONCLUSIONS: Insulin-resistant ob/ob cardiomyocytes showed decreases in DAG-mediated NSCC, which were accompanied by decreased TRPC3 expression and defective insulin-mediated trafficking of this protein.     

Simvastatin but not ezetimibe reduces sympathetic activity despite similar reductions in cholesterol levels

The relationship between the sympatholytic effects of statins and their lipid-lowering activity remains unclear. Ezetimibe lowers cholesterol, but its sympatholytic activity is unknown. The purpose of study was to compare the influence of equipotent doses of simvastatin and ezetimibe on sympathetic activity. This randomized double-blinded study was performed in 22 hypertensive patients (age, 45.6 ± 2.2 years; female/male, 2/20) with untreated hypercholesterolemia. The subjects were administered 20 mg/d of simvastatin (n = 11) or 20 mg/d of ezetimibe (n = 11) for 6 weeks. Pre- and post-treatment measurements of muscle sympathetic nerve activity (MSNA), baroreceptor control of heart rate (baroreflex sensitivity), and impedance cardiography were recorded. Simvastatin and ezetimibe produced similar reductions of total (−58.0 ± 23.4 vs. −45.2 ± 17.2 mg/dL; P = .15, respectively) and low-density lipoprotein cholesterol (−52.6 ± 20.9 vs. −37.9 ± 17.6 mg/dL; P = .09, respectively). There was a significant difference in the effect of simvastatin and ezetimibe on muscle sympathetic nerve activity (−8.5 ± 5.1 vs. −0.7 ± 3.5 bursts/min; P = .0005). Simvastatin improved baroreflex sensitivity as compared with ezetimibe (10.0 ± 14.3 vs. −2.8 ± 6.1 ms/mm Hg; P = .01). There was no difference in the effect of both treatments on blood pressure, heart rate, cardiac output, stroke volume, total peripheral resistance, high-density lipoprotein, and triglycerides. Simvastatin reduced sympathetic activity via lipid-independent mechanisms, but ezetimibe exerts no sympatholytic effects.     

Hyperoxia, but not thoracic X-irradiation, potentiates bleomycin- and cyclophosphamide-induced lung damage in mice

The intraperitoneal administration of cyclophosphamide or bleomycin to BALB/c mice resulted in lung cell damage followed by cellular proliferation, which was quantitated by measuring the increase in thymidine incorporation into pulmonary DNA. We have previously shown that administration of the antioxidant butylated hydroxytoluene produces lung damage that can be potentiated by both hyperoxia and thoracic X-irradiation. In the present study we show that hyperoxic exposure also potentiates bleomycin- and cyclophosphamide-induced acute lung damage. However, thoracic X-irradiation does not potentiate bleomycin- and cyclophosphamide-induced lung toxicity.