Glutamate reduces secretion of l-serine in astrocytes isolated from stroke-prone spontaneously hypertensive rats

In the CNS, l-serine (l-Ser) plays an essential role in neuronal survival by evoking a variety of biological responses in glial cells. Initially, we examined whether glutamate, hydrogen peroxide (H(2)O(2)), interleukin-1 (IL-1) beta, and sodium nitroprusside (SNP) induce the secretion of l-Ser in astrocytes isolated from Wistar Kyoto rats (WKY). The secretion of l-Ser was significantly induced with glutamate and SNP in cultured astrocytes. Next, to gain insight into the involvement of l-Ser in glutamate-induced neuroprotection, we compared the secretion of l-Ser in astrocytes isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive rats, WKY. We also examined the mRNA expression of the enzyme that produces l-Ser, 3-phosphoglycerate dehydrogenase (PHGDH), and a neural amino acid transporter, ASCT1, in the cultured astrocytes. A dose-dependent study of glutamate in astrocytes of SHRSP indicated differences in the secretion of l-Ser, and gene expression of PHGDH and ASCT1, compared with levels in the WKY astrocytes. We demonstrated that both the secretion and the gene expression were significantly attenuated in glutamate-treated astrocytes from SHRSP. Cerebral ischemia in SHRSP induced a massive efflux of glutamate, causing delayed neuronal death in region CA1 of the hippocampus. The results suggest that the attenuated secretion of l-Ser in astrocytes is involved in neuronal vulnerability and survival in SHRSP during the production of glutamate, as the secretion of l-Ser, which is stimulated by glutamate, is closely related to the protective effect against glutamate-mediated neurotoxicity. We conclude that glutamate and SNP up-regulate the secretion of l-Ser in primary astrocytes. Secretion of l-Ser is regulated in astrocytes in response to glutamate and nitric oxide and may correspond to the level of l-Ser needed for neuronal survival during brain insults such as ischemic stroke in SHRSP.     

A2B adenosine receptors induce IL-19 from bronchial epithelial cells, resulting in TNF-alpha increase

Adenosine is a signaling nucleoside that has been proposed to contribute to the pathogenesis of asthma and chronic obstructive pulmonary disease. Previous studies suggest that adenosine might play an important role in modulating levels of inflammatory mediators in the lung. Because airway epithelium is an important cellular source of inflammatory mediators, the objective of the present study was to determine whether adenosine affects the expression and release of inflammatory cytokines from human bronchial epithelial cells (HBECs). Among the four subtypes of adenosine receptors, the A(2B) receptor was expressed at the highest level. 5'-(N-ethylcarboxamido)-adenosine (NECA), a stable analog of adenosine, increased the release of IL-19 by 4.6- +/- 1.1-fold. A selective antagonist of the A(2B) receptor, CVT-6694, attenuated this effect of NECA. The amount of IL-19 released from HBEC was sufficient to activate a human monocytic cell line (THP-1) and increase the release of TNF-alpha. Furthermore, TNF-alpha was found to upregulate A(2B) receptor expression in HBECs by 3.1- +/- 0.3-fold. Hence, these data indicate that NECA increases the release of IL-19 from HBECs via activation of A(2B) receptors, and IL-19 in turn activates human monocytes to release TNF-alpha, which upregulates A(2B) receptor expression in HBECs. The results of this study suggest that there is a novel pathway whereby adenosine can initiate and amplify an inflammatory response which might be important in pathogenesis of inflammatory lung diseases.     

Modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the A(2B) receptor

Adenosine triphosphate has previously been shown to induce semi-mature human monocyte-derived dendritic cells (DC). These are characterized by the up-regulation of co-stimulatory molecules, the inhibition of IL-12 and the up-regulation of some genes involved in immune tolerance, such as thrombospondin-1 and indoleamine 2,3-dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y(11) receptor; since there is no functional P2Y(11) gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5'-(3-thiotriphosphate) and adenosine inhibited the production of IL-12p70 by bone marrow-derived DC (BMDC). These inhibitions were relieved by 8-p-sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A(2B) (-/-) BMDC indicated the involvement of the A(2B) receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5'-(N-ethylcarboxamido) adenosine (NECA, the most potent A(2B) receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin-1 and vascular endothelial growth factor were up-regulated whereas CCL2 and CCL12 were down-regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A(2B) receptor, and will possibly lead to an impairment of Th1 response or tolerance.     

Superoxide dismutase reduces the impairment of endothelium-dependent relaxation in the spontaneously hypertensive rat aorta.

The involvement of the superoxide anion in endothelium-dependent relaxation (EDR) was examined in noradrenaline-contracted aortic smooth muscle preparations isolated from normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Acetylcholine (ACh, 10(-9)-10(-5) M) induced EDR in both WKY and SHRSP preparations in a concentration-dependent manner, but with a significantly smaller amplitude in those from SHRSP than in those from WKY. The ACh-induced EDR was inhibited by N(omega)-nitro-L-arginine (L-NOARG), in a concentration-dependent manner, both in WKY and SHRSP. The EDR produced in WKY in the presence of 3 x 10(-6) M L-NOARG was similar in magnitude to that produced in SHRSP in the absence of L-NOARG. Superoxide dismutase (SOD, 300 units/ml) increased the amplitude of EDR in SHRSP but not in WKY, with no alteration of the threshold or of the maximal amplitude. The maximal amplitude of EDR produced in SHRSP in the presence of SOD was still smaller than that in WKY. In WKY, a possible involvement of superoxide in the EDR was examined in aortae whose EDR was partially inhibited by treatment with a subthreshold concentration (3 x 10 (-6) M) of L-NOARG. In the L-NOARG-conditioned aorta, the reduced EDR was partially but significantly recovered by SOD. These results suggest that the impaired EDR in aortae of SHRSP may be causally related to a higher production of superoxide. The L-NOARG-induced inhibition of EDR in WKY may be produced, in part, by the reduction of effective NO due to its destruction by superoxide.     

Electrophysiological evidence for adenosine receptors on astrocytes of cultured rat central nervous system

The actions of adenosine, R-N6-phenylisopropyladenosine (R-PIA) and N-(ethyl)carboxamidoadenosine (NECA) were tested on the membrane potential of astrocytes of cultured rat spinal cord and cerebellum. All 3 compounds hyperpolarized the majority of astrocytes studied. A considerable number of cells did, however, not respond to adenosine and its analogues, suggesting that only a certain type of astrocyte possesses adenosine receptors. The hyperpolarizations by adenosine, R-PIA and NECA were reversibly blocked by their antagonist 8-phenyltheophylline indicating that these compounds activate specific adenosine receptors. In agreement with biochemical and autoradiographic binding studies, our electrophysiological data strongly suggest the existence of adenosine receptors on astrocytes.     

Differential regulation of the nitric oxide-cGMP pathway exacerbates postischaemic heart injury in stroke-prone hypertensive rats

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO-cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia-reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca(2+)-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca(2+)-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca(2+)-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca(2+)-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.     

Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin

Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.     

Neuronal vulnerability of stroke-prone spontaneously hypertensive rats to ischemia and its prevention with antioxidants such as vitamin E

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension, and more than 95% of them die of cerebral stroke. Hypoxic stimulation followed by oxygen reperfusion induces neuronal damage in both normotensive Wistar Kyoto/Izm (WKY/Izm) and SHRSP/Izm rats, and the percentage of neurons that undergo apoptosis during hypoxia-reperfusion is markedly higher in SHRSP/Izm rats than in WKY/Izm rats. The biochemical characteristics of the SHRSP/Izm rats, unlike those of WKY/Izm rats, might act as a factor in the stroke proneness of SHRSP/Izm rats. In the hippocampus, the formation of hydroxyl radicals and the cerebral blood flow-independent formation of nitric oxide (NO) were strongly increased after reperfusion in SHRSP/Izm rats, and the neuronal expression of the thioredoxin and Bcl-2 genes was significantly decreased in the SHRSP/Izm rats compared with the WKY/Izm rats. On the other hand, the effects of antioxidants against neuronal death associated with cerebral ischemia-reperfusion were stronger in the SHRSP/Izm rats, in which the addition of vitamin E or ebselen almost completely inhibited neuronal death. Namely, the addition of 100 μg/ml of vitamin E under hypoxia/reoxygenation (H/R) conditions completely inhibited WKY and SHRSP/Izm neuronal death. Vitamin E exerts a marked inhibitory effect against neuronal damage via its incorporation into mitochondrial membranes, where it captures reactive oxygen and free radicals. The susceptibility of neurons to apoptosis in SHRSP/Izm rats is partly due to an insufficiency of mitochondrial redox regulation and apoptosis-inhibitory proteins. In this review, we describe the neuronal vulnerability of SHRSP/Izm rats induced by cerebral ischemia and the effects of antioxidants such as vitamin E.     

Unaltered caffeine-induced relaxation in the aorta of stroke-prone spontaneously hypertensive rats (SHRSP).

Caffeine-induced relaxation was studied in aortic segments from Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Although acetylcholine-induced endothelium-dependent relaxation was impaired in preparations from SHRSP, the relaxation induced by caffeine was identical in both groups. In addition, caffeine-induced relaxation was not affected by removal of the endothelium in either group. The relaxation induced by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), a membrane-permeable analog of adenosine 3':5'-cyclic monophosphate (cAMP), was identical in both groups. No significant difference was observed in the increase in cAMP content induced by caffeine in the aortic smooth muscle between the groups, although the basal content was significantly higher in preparations from SHRSP. These results suggest that the relaxation induced by caffeine in these preparations is brought about by its direct effect on smooth muscle and that the response of the smooth muscle to caffeine, including cAMP production, is not altered in preparations from SHRSP compared with those from WKY.     

Activation of mast cells by immunoglobulin E-receptor cross-linkage,but not through adenosine receptors,induces A1 expression and promotes survival

BACKGROUND: Mast cells are a potent source of mediators that regulate the inflammatory response in allergy and asthma. Mast cells can be activated through different receptors, for example, via cross-linkage of the high-affinity IgE receptor (Fc epsilon RI) and by adenosine acting on specific receptors. We have recently described mast cell survival of an IgE receptor activation by up-regulation of the anti-apoptotic gene A1. OBJECTIVE: To compare mast cell survival and expression of A1 after activation through the Fc epsilon RI and by an adenosine agonist. METHODS: Bone marrow-derived, cultured mouse mast cells (BMCMC) were activated either with IgE+antigen or with the adenosine receptor agonist 5'-N-ethylcarboxamido adenosine (NECA). Release of beta-hexosaminidase, cell viability, phosphorylation of Akt and IkB-alpha, and expression of pro-survival and pro-apoptotic genes were measured after activation. RESULTS: Activation of BMCMC with NECA caused the release of beta-hexosaminidase, although to a lesser extent than after Fc epsilon RI activation (33% and 98%, respectively). Activation by both NECA and Fc epsilon RI stimulated phosphorylation of Akt (Ser473 and Thr308) and IkB-alpha (Ser32), both of which are implicated in the regulation of cell survival. However, only cells that were activated through Fc epsilon RI, but not by NECA, expressed A1 and exhibited an increased survival rate compared to the control. CONCLUSION: These results show that adenosine receptor activation of BMCMC does not induce the same survival programme in mast cells as does activation through Fc epsilon RI. These findings may be important for understanding the role that mast cells play in asthma provoked by different stimuli.     

A monoclonal anti-idiotypic 'internal image' antibody that recognizes the A1 adenosine receptor potentiates the alpha 1-adrenergic activation of phospholipase C in primary cultures of mouse striatal astrocytes.

To determine which subtype of adenosine receptor mediates the potentiating effect of 2-chloroadenosine on the noradrenaline-induced inositol-phosphate formation, we used the monoclonal anti-idiotypic antibody AA1 that acts as an 'internal image' of adenosine and specifically recognizes the A1 adenosine receptor. In cultured mouse striatal astrocytes, AA1 increased the noradrenaline-evoked inositol phosphate (IP) accumulation, thus demonstrating a biological activity of an anti-idiotypic antibody. This effect was inhibited by PACPX, a selective A1 antagonist. Inhibitors of phospholipase A2 activity prevented the potentiation. These results establish the involvement of A1 adenosine receptors in the modulation of phospholipase C activity.     

Adenosine analogues stimulate cyclic AMP formation in rabbit cerebral microvessels via adenosine A2-receptors

This study has examined the accumulation of cyclic AMP in microvessels from rabbit and feline cerebral cortex induced by a series of adenosine analogues to determine the type of receptor involved. The conversion of tritium labelled adenine nucleotides to [3H]cyclic AMP was determined in [3H]adenine labelled microvessels in the presence of an inhibitor of cyclic AMP hydrolysis, rolipram (30 microM). In microvessels from both cats and rabbits two adenosine analogues, N-5'-ethylcarboxamido adenosine (NECA) and L (S)-phenylisopropyladenosine (PIA), stimulated cyclic AMP accumulation. The response was larger and more reproducible in rabbits than in cats. In rabbit cerebral microvessels the order of potency as stimulator of cyclic AMP accumulation was NECA greater than 2-chloroadenosine greater than L-PIA greater than cyclohexyladenosine (CHA) greater than D-PIA. This order of potency defines the receptor involved as being of the A2 subtype. Although the maximal response to CHA appeared to be lower than that to NECA, CHA did not inhibit the response to NECA, suggesting that it is not a classical partial agonist. In the presence of the adenylate cyclase stimulating compound forskolin (I microM) NECA was more active than in its absence (close to 30-fold increase in EC50) and also produced a maximal effect six times higher. The maximal responses to PIA and CHA increased proportionally in the presence of forskolin. These results show that rabbit cerebral microvessels possess adenosine receptors of the A2 subtype capable of stimulating the formation of cyclic AMP. The functional significance of such receptors is not known, but may be related to regulation of vascular permeability.     

Theophylline inhibits the differentiation of human monocyte into dendritic cell potentially via adenosine receptor antagonism

Background Theophylline has an anti‐inflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airways. Dendritic cells (DCs) are professional antigen‐presenting cells, capable of priming naïve T cells, and play key roles in the activation of immune responses in asthma. Objective The purpose of this study was to investigate the effects of theophylline on human monocyte differentiation into DCs and whether this involved antagonism of adenosine receptors. Methods Peripheral human blood monocytes were cultured in the presence of granulocyte/macrophage‐colony stimulating factor and IL‐4 to induce DC differentiation. The cells were incubated with theophylline, KF17837 (a selective A2a receptor antagonist) and enprofylline (A2b receptor antagonist) and co‐incubated with selective adenosine A1 and A2a receptor agonists, a phosphodiesterase inhibitor (rolipram) and adenosine deaminase (ADA) to determine their effects on DC differentiation. In addition, depletion of adenosine receptors by small interfering RNA (siRNA) was also examined. Results Monocytes differentiated into myeloid DCs in the culture system. The number of DCs was remarkably reduced by 60–70% when theophylline was administered at a therapeutic concentration. This effect was concentration‐dependently exacerbated, was partly mediated by cellular apoptosis and was effectively reversed by the addition of the A1 agonists [2‐chloro‐N⁶‐cyclopentyladenosin, N⁶‐cyclohexyladenosine, and N‐ethylcarboxamidoadenosine (NECA)] or the A2a agonist (CGS‐21680, NECA). The depletion of the adenosine A1 receptor by siRNA and addition of ADA remarkably reduced DC differentiation. Meanwhile, both enprofylline and rolipram had little effect. Conclusion Our findings suggest that the adenosine A1 (and possibly coordinated with A2a) receptors contribute to DC differentiation and survival. These findings provide further evidence that theophylline has an anti‐inflammatory action in bronchial asthma.     

Potential role of glial cell line-derived neurotrophic factor receptors in Müller glial cells during light-induced retinal degeneration

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and their receptors (GFRalpha1, GFRalpha2 and Ret) play an important role in the survival of neurons in the central and peripheral nervous system. For example, GDNF as well as other trophic factors promotes photoreceptor survival during retinal degeneration. Recent studies have proposed that part of neurotophic rescue of photoreceptors may be indirect, mediated by interaction of the neurotrophic factors with other cell types, that in turn release secondary factors that act directly on photoreceptors. In the present study, we examined the GDNF receptor expression in control and light-damaged retina, and found that GFRalpha2 protein is upregulated in retina-specific Müller glial cells during photoreceptor degeneration. We also examined the effect of GDNF or NTN on cultured Müller cells. Exogenous GDNF increased brain-derived neurotrophic factor, basic fibroblast growth factor and GDNF, but not NTN mRNA production. On the other hand, NTN increased NTN, but not GDNF mRNA production in cultured Müller cells. These observations suggest that GDNF, NTN and their receptors are involved in the regulation of trophic factor production in retinal glial cells, and that functional glia-neuron network may utilize GDNF family for the protection of neural cells during retinal degeneration.     

Adenosine signalling in T-cell activation favours development of IL-17 positive cells with suppressive properties

Evidence suggests that the anti-inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (T_reg)/helper (Th17) T-cell balance in favour of T_reg. Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T-cells. Proliferation, phenotype and cytokine production of stimulated T-cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan-adenosine agonist 5′-N-ethylcarboxamidoadenosine (NECA) skews human CD3⁺ T-cell responses towards non-inflammatory Th17 cells. Addition of NECA during T-cell activation increased the development of IL-17⁺ cells with a CD4⁺ RORγt⁺ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non-inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell-like molecular signature and induced surface expression of the adenosine-producing ectoenzymes CD39 and CD73. Thus, T-cells cultured under Th17-inducing conditions together with NECA were capable of suppressing responder T-cells. Finally, genome-wide gene expression profiling revealed metabolic quiescence previously associated with non-pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non-inflammatory Th17 cells in human T-cell differentiation, potentially through regulation of metabolic pathways.     

Noradrenergic hyperinnervation may inhibit necrosis of coronary arterial smooth muscle cells in stroke-prone spontaneously hypertensive rats

Noradrenergic (NA) nerve fibre distribution and vascular smooth muscle morphology were investigated in the coronary artery of stroke-prone spontaneously hypertensive rats (SHRSP). Fluorescent NA nerve fibres of SHRSP aged 10, 30, 60, 90 and 180 days were examined by the glyoxylic acid method and compared with those of age-matched normotensive Wistar Kyoto (WKY) rats. The distribution densities of NA nerve fibres were measured by quantitative image analysis using the Interactive Bildanalyse System. The densities of NA nerve fibres of the left coronary artery of SHRSP were significantly higher than those of WKY rats at all ages examined. NA hyperinnervation in the coronary artery of SHRSP may be caused by the hyperfunction of the stellate ganglia which innervate the coronary arteries. Scanning electron microscopy observations showed that the surface of smooth muscle cells of the left coronary artery in SHRSP was smooth and similar to that of WKY rats at 120 days of age, but was slightly modified by more invaginations and projections than that in WKY rats at 180 days of age. No necrotic cells, however, were found in SHRSP. By transmission electron microscopy the smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and surrounded by many layers of basal laminalike material, but no necrotic cells were found. We suggest that NA hyperinnervation protects the vascular smooth muscle cells from necrosis in the coronary artery of SHRSP by a trophic effect mediated by NA nerve fibres.