Presence of specific bradykinin receptors in arterial and venous smooth muscle

The relationship between bradykinin action and its concentration was examined on isolated rings of the rabbit aorta, femoral artery, jugular vein and on isolated strips of the rat portal vein. The sensitivity of femoral artery and portal vein smooth muscles to bradykinin was disclosed. Venous smooth muscles were more sensitive to bradykinin as compared with arterial smooth muscles. Dissociation constants for the rabbit aorta, femoral artery, jugular vein and for the rat portal vein were 3.98 X 10(-6), 6.3 X 10(-6), 1.26 X 10(-7), and 7.6 X 10(-9)M, respectively. Effects of endogenous bradykinin in vivo might result from its primary action on the venous smooth muscle, action on the arterial smooth muscle and veno-arterial interactions.     

Potential role of the neuropeptide CGRP in the induction of differentiation of rat hepatic portal vein wall

The media of the rat hepatic portal vein is composed of an internal circular muscular layer (CL) and an external longitudinal muscular layer (LL). These two perpendicular layers differentiate progressively from mesenchymal cells within the first month after birth. In this paper, we studied the development of calcitonin gene-related peptide (CGRP) innervation during post-natal differentiation of the vessel. We show that CGRP innervation is already present around the vessel at birth in the future adventitia but far from the lumen of the vessel. Progressively, CGRP immunoreactive fibers reached first LL then CL. CL by itself become only innervated at day 14 after birth. This corresponds to the time at which thick filaments (myosin) are visible in electron microscopy and desmin visualisable by immunocytochemistry. Furthermore, we provide evidence by autoradiography, that binding sites for CGRP are transiently expressed on the portal vein media at day 1 and 14 after birth. Vascular smooth muscle cells were transfected with constructs containing promoters for desmin or smooth muscle myosin heavy chain (smMHC). CGRP treatment of the cells significantly increased the expression of smMHC. Overall these results suggest that CGRP can potentially influence the differentiation of smooth muscle cells from the vessel wall.     

Norepinephrine, monoamine oxidase, and acetylcholinesterase in the rat jugular vein compared with other blood vessels

The observation that the rat jugular vein relaxed in response to norepinephrine but not to field stimulation prompted us to evaluate the extent of innervation in this tissue. The norepinephrine concentration in the jugular vein was about 10% of that in the mesenteric artery and vein. The low levels of norepinephrine were not due to higher monoamine oxidase activity relative to the enzyme activity in other blood vessels. In the jugular vein, as in heart and brain, serotonin was preferred substrate for monoamine oxidase whereas in the femoral vein, mesenteric vein, and mesenteric artery, phenylethylamine oxidation was greater. Based on kinetic and inhibitory studies with LY51641, a selective type A inhibitor, monoamine oxidase activity was not found to be uniform throughout the cardiovascular system. In addition to low levels of norepinephrine, acetylcholinesterase activity in the jugular vein was only 5 and 13% of the activity in the portal vein and mesenteric artery, respectively. Thus, we provide strong evidence that our inability to generate a response to field stimulation in the rat jugular vein results from the lack of functional innervation in this tissue. This information adds to the usefulness of this preparation for comparative studies of agents acting on the smooth muscle without the added complication of neuronal uptake mechanisms.     

Modulation by GABA of neuroplasticity in the central and peripheral nervous system

Apart from being a prominent (inhibitory) neurotransmitter that is widely distributed in the central and peripheral nervous system, -aminobutyric acid (GABA) has turned out to exert trophic actions. In this manner GABA may modulate the neuroplastic capacity of neurons and neuron-like cells under various conditions in situ and in vitro. In the superior cervical ganglion (SCG) of adult rat, GABA induces the formation of free postsynaptic-like densities on the dendrites of principal neurons and enables implanted foreign (cholinergic) nerves to establish functional synaptic contacts, even while preexisting connections of the preganglionic axons persist. Apart from postsynaptic effects, GABA inhibits acetylcholine release from preganglionic nerve terminals and changes, at least transiently, the neurochemical markers of cholinergic innervation (acetylcholinesterase and nicotinic receptors). In murine neuroblastoma cells in vitro, GABA induces electron microscopic changes, which are similar in principle to those seen in the SCG. Both neuroplastic effects of GABA, in situ and in vitro, could be mimicked by sodium bromide, a hyperpolarizing agent. In addition, evidence is available that GABA via A- and/or B-receptors may exert direct trophic actions. The regulation of both types of trophic actions (direct, receptor-mediated vs. indirect, bioelectric activity dependent) is discussed.Special issue dedicated to Dr. Claude Baxter.     

Electrophysiological analysis of the action of kavinton on the smooth muscles

Cavinton at a concentration of 10(-7)-10(-5) M was found to have a dose-dependent relaxing effect on bovine cerebral artery smooth muscles, without changing the resting potential and membrane resistance. Smooth muscles of the rabbit portal vein and guinea-pig taenia coli were insensitive to low cavinton concentrations. The results are consistent with the hypothesis that relaxing action of cavinton is due to the blocking of Ca2+ ions influx into the cells of cerebral artery through receptor-operated calcium channels. At higher concentrations (exceeding 10(-5) M) cavinton exerts nonspecific influence on the smooth muscles under study, inhibiting their excitability and decreasing membrane resistance resulting in the attenuation of tetanic contractions in the smooth muscles of the portal vein and taenia coli.     

Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle

The Ca(2+)-independent acceleration of dephosphorylation of the regulatory light chain of smooth muscle myosin and relaxation of smooth muscle by telokin are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al. (1998) J. Biol. Chem. 273, 11362-113691. The purpose of this study was to determine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by different kinases. The in vivo site(s) of phosphorylation of telokin were determined in rabbit smooth muscles of longitudinal ileum and portal vein. Following stimulation of ileum with forskolin (20 microM) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit portal vein telokin was phosphorylated on both Ser-13 and -19 as a result of forskolin and GTPgammaS stimulation in vivo. Point mutation of Ser-13 (to Ala or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases.     

The role of osmotic factors in the effect of mineral water Naftusia on smooth muscles of the portal vein

The effect of the Krebs solution tonicity on the electrical and mechanical activity of smooth muscles has been studied while adding 1-5% volume of mineral water Naftusya, its artificial salt analog, and distilled water into the above solution. It is shown that effects observed are induced by the medium tonicity changes rather than by biologically active components of the mineral water. The smooth muscle cells of the rat portal vein are sensitive osmometers which affect hypotonic Krebs solution by 1%.     

Developmental expression of dystrophin on the rat myocardial cell membrane

The Duchenne muscular dystrophy gene product, dystrophin, is expressed on the cell membrane of skeletal and cardiac muscles. We examined the developmental time course of dystrophin expression in the rat ventricular myocardium. Dystrophin was immunohistochemically undetectable on the 15th embryonic day, but a small amount was expressed on the cell membrane on the 17th embryonic day. The amount increased during the perinatal period reaching adult level at 2 weeks after birth. These findings were supported by immunoblot analysis. Chemical sympathectomy in newborn rats by 6-hydroxydopamine (prevention of innervation) had no detectable influence on myocardial dystrophin expression at 2 weeks after birth. Our results show that dystrophin lining on the rat myocardial cell membrane increases during the perinatal period, and that this process is little influenced by the development of sympathetic innervation.     

Developmental expression of dystrophin on the rat myocardial cell membrane

Summary The Duchenne muscular dystrophy gene product, dystrophin, is expressed on the cell membrane of skeletal and cardiac muscles. We examined the developmental time course of dystrophin expression in the rat ventricular myocardium. Dystrophin was immunohistochemically undetectable on the 15th embryonic day, but a small amount was expressed on the cell membrane on the 17th embryonic day. The amount increased during the perinatal period reaching adult level at 2 weeks after birth. These findings were supported by immunoblot analysis. Chemical sympathectomy in newborn rats by 6-hydroxydopamine (prevention of innervation) had no detectable influence on myocardial dystrophin expression at 2 weeks after birth. Our results show that dystrophin lining on the rat myocardial cell membrane increases during the perinatal period, and that this process is little influenced by the development of sympathetic innervation.     

Smooth muscle function of the portal vein in spontaneously hypertensive rats

Increased contractility and adrenoreactivity of the portal vein smooth muscles were revealed in spontaneously hypertensive rats (SHR) only at the early stage of the disease. In stable hypertension the changes were milder both at the early and chronic stages. Portal vein smooth muscles were capable of contracting in low-calcium medium, which suggests a membrane defect in the smooth muscles of animals with arterial hypertension.     

Tris(hydroxymethyl)aminomethane inhibits calcium uptake in vascular smooth muscle.

This report demonstrates that the commonly used buffering agent Tris(hydroxymethyl)aminomethane (Tris) in concentrations of 5 and 30 mM inhibits calcium (Ca2+) uptake in both rat aortic and portal venous smooth muscle. The data indicates that total exchangeable Ca2+ in portal vein is reduced by about 35% in 5 or 30 mM Tris, while the intracellular exchangeable Ca2+ is not significantly altered. On the other hand, in aortic smooth muscle, while 30 mM Tris reduces total exchangeable Ca2+ by about 20%, intracellular Ca2+ is reduced by 44% in 5 mM Tris and by 55% in 30 mM Tris. The present studies, thus, reveal that Tris exerts significant inhibitory effects on exchangeability and transmembrane movement of Ca2+ in at least two different types of smooth muscle.     

Angiotensin II-induced delayed stimulation of phospholipase C gamma1 requires activation of both phosphatidylinositol 3-kinase gamma and tyrosine kinase in vascular myocytes

In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K). We investigated the time-dependent effects of AII on both phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3-5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kgamma and that this isoform was involved in the AII-induced stimulation of InsPs accumulation. NA-induced InsPs accumulation depended on PLCbeta1 activation whereas AII-induced InsPs accumulation depended on PLCgamma1 activation. AII-induced PLCgamma1 activation required both tyrosine kinase and PI3Kgamma since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti-PI3Kgamma antibody abolished AII-induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCgamma1 was only detected for long-lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kgamma and tyrosine kinase is a prerequisite for AII-induced stimulation of PLCgamma1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long-lasting contraction induced by AII.     

Study of Mechanisms of Relaxation in Rat Blood Vessels Differing by Density and Innervation Type

The work deals with a study of physiological significance of relaxation mechanisms initiated by stimulation of -adrenoreceptors and NO-dependent pathways as well as of participation of cyclic nucleotide signal pathways in dilatation of rat blood vessels with different density and type of nervous regulation: of thoracic aorta with poorly developed adrenergic vasoconstrictional innervation, of tail artery with well-developed adrenergic vasoconstrictional innervation, and of portal vein with adrenergic and cholinergic vasoconstrictional innervation. Isometric contraction of 1-mm wide vascular rings produced by electrostimulation or action of exogenous selective antagonists (phenylephrine for ₁-adrenoreceptors of all three vessels and 5-methylformetide for muscarinic cholinergic receptors of portal vein) was recorded using a laboratory-made highly sensitive device. From relaxants, isoproterenol, sodium nitroprusside, forskolin, and isobutylmethylxanthine were used. As a result of the performed study with use of substances affecting various chains of processes providing relaxation of smooth muscle contractile apparatus of blood vessels with different degree of development of nervous regulation, we have obtained data that indicate heterogeneity of intracellular mechanisms of transmission of external signal realizing the contractionndash;relaxation cycle in these vessels.     

Chronic treatment of rats with D-600 causes a compensatory decrease in the calcium requirement for contractility of vascular smooth and cardiac muscles

We studied the effects of chronic hypotensive treatment of normotensive Wistar rats (NWR) with methoxyverapamil (D-600) and hydralazine on in vitro contractile response of aortic strips, portal vein strips, and Langendorff-perfused hearts in normal (2.5 mM) and low (0.2 mM) calcium (Ca). Portal vein strips from rats treated with D-600, compared with the same strips from control and hydralazine-treated rats, developed greater spontaneous contractile activity in normal Ca and retained greater responses to norepinephrine (NE) and 80 mM K in low Ca. Aortic strips from all three groups of rats retained similar responses to NE and K in low Ca. Hearts from D-600-treated rats produced less intraventricular pressure (IVP) to isoproterenol (ISO) than hearts from control and hydralazine-treated rats in normal Ca but greater IVP to ISO than hearts from the other two groups of rats in low Ca. Thus, chronic treatment of NWR with D-600 but not with hydralazine resulted in the reduction of Ca requirement for contractile activities of the portal vein and the myocardium.     

An improved glyoxylic acid technique for the histochemical localization of catecholamines in brown adipose tissue

Summary A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.     

Postnatal ontogenic development of the adrenergic innervation pattern in the rat portal vein

Summary The postnatal development of the adrenergic innervation pattern in the rat portal vein has been studied with the histochemical fluorescence method of Hillarp and Falck.Stretch preparations and transverse freeze-dried sections of intact portal veins were studied from rats during the first 5 weeks of life and from adult rats. Orientation of undifferentiated smooth muscle cells into two layers was observed at 4 days of age. Dominance of the thick outer longitudinal muscle layer was apparent at two weeks of age. A terminal adrenergic nerve plexus with some varicosities was restricted outside the media at the end of the first week. Ingrowth of penetrating non-terminal adrenergic nerve fibers through the longitudinal muscle layer occurred during the second week of age when the main terminal nerve plexus was developing between the two muscle layers. After 3 weeks of age the adult pattern of a two-dimensional adrenergic plexus between the muscle was established. In the adult rat pharmacological treatment with nialamide and noradrenaline revealed the thin, penetrating non-terminal adrenergic nerve fibers in the longitudinal muscle layer which were poorly visible otherwise.The present observations strongly indicate that the main adrenergic plexus between the two muscle layers emanates directly from the outer axonal plexus. These findings are discussed regarding possible trophic interactions between ingrowing sympathetic adrenergic vasomotor nerves and maturing vascular smooth muscle.Supported by grants from the Swedish Medical Research Council (grants No. 14X-2207, O4P-4173, 3884), Magn. Bergwall's Foundation, G. & M. Lindgren's Foundation, the Medical Faculty of University of Göteborg. The technical assistance of Miss Serney Bööj, Mr. Pär-Anders Larsson and Miss Ann Kjellstedt is gratefully acknowledged     

Pentobarbital sodium inhibits calcium uptake in vascular smooth muscle

This report demonstrates that the commonly used anesthetic agent, pentobarbital sodium, in concentrations of 1 · 10^?4 to 2 · 10^?3 M inhibits calcium (Ca²⁺) uptake in both rat aortic and portal venous smooth muscle. The data indicate that total exchangeable Ca²⁺ in portal vein is reduced by about 15% in 1 · 10^?4 M pentobarbital sodium, while the intracellular exchangeable Ca²⁺ is reduced by 24%. On the other hand, in aortic smooth muscle, while 5–20 · 10^?4 M pentobarbital sodium reduces total exchangeable Ca²⁺ by about 15%, intracellular Ca²⁺ is reduced by 22% in 5 · 10^?4 M pentobarbital sodium and by 38% in 2 · 10^?3 M pentobarbital sodium. The present studies thus reveal that concentrations of pentobarbital sodium known to be present during induction of surgical anesthesia can exert significant inhibitory effects on exchangeability and transmembrane movement of Ca²⁺ in at least two different types of blood vessels.     

Sympathetic neural influences on muscle blood flow in rats during submaximal exercise

These experiments were designed to estimate the involvement of the sympathetic innervation in regulation of hindlimb muscle blood flow distribution among and within muscles during submaximal locomotory exercise in rats. Blood flows to 32 hindlimb muscles and 13 other selected tissues were measured using the radiolabeled microsphere technique, before exercise and at 0.5, 2, 5, and 15 min of treadmill exercise at 15 m/min. The two groups of rats studied were 1) intact control, and 2) acutely sympathectomized (hindlimb sympathectomy accomplished by bilateral section of the lumbar sympathetic chain and its connections to the spinal cord at L2-L3). There were no differences in total hindlimb muscle blood flow among the two groups during preexercise or at 30 s or 2 min of exercise. However, flow was higher in eight individual muscles at 2 min of exercise in the sympathectomized rats. At 5 and 15 min of exercise there was higher total hindlimb muscle blood flow in the denervated group compared with control. These differences were also present in many individual muscles. Our results suggest that 1) sympathetic nerves do not exert a net influence on the initial elevations in muscle blood flow at the beginning of exercise, 2) sympathetic nerves are involved in regulating muscle blood flow during steady-state submaximal exercise in conscious rats, and 3) these changes are seen in muscles of all fiber types.     

Norepinephrine protects against apoptosis of mesenchymal stem cells induced by high glucose

In diabetes, the number of bone mesenchymal stem cells (MSCs) decreases and their differentiation is impaired. However, the exact mechanism is unclear. Patients with diabetes often experience sympathetic nerve injury. Norepinephrine (NE), a major mediator of the sympathetic nervous system, influences rat MSC migration in culture and in vivo. The present study aimed to investigate the effect of NE on MSCs under high glucose conditions; therefore MSCs were treated with high glucose and NE. High glucose-induced MSCs apoptosis, which was reversed by NE. To verify the effect of NE, mice underwent sympathectomy and were used to establish a diabetic model. Diabetic mice with sympathectomy had a higher apoptosis rate and higher levels of reactive oxygen species in their bone marrow-derived cells than diabetic mice without sympathectomy. High glucose inhibited p-AKT production and B-Cell CLL/Lymphoma 2 expression, and promoted BAX and caspase-3 expression. NE reversed these effects of high glucose. An AKT inhibitor enhanced the effects of high glucose. Thus, NE had a protective effect on MSC apoptosis induced by high glucose, possibly via the AKT/BCL-2 pathway.