Higher Ca2+ -sensitivity of arterial contraction in 1-week-old rats is due to a greater Rho-kinase activity.

AIM: During early post-natal development, arterial contraction depends less on Ca2+ -signalling pathways but more on changes in Ca2+ -sensitivity compared to adult animals. Whether this difference is related to Rho-kinase, one of the major players affecting Ca2+ -sensitivity, is unknown for intact vessels. Thus, we tested the hypothesis that Rho-kinase critically contributes to the higher Ca2+ -sensitivity of contraction in intact arteries of 1-week-old rats. METHODS: We studied 1-week-old, 4- to 5-week-old and 10- to 12-week-old rats performing isometric myography, Ca2+ -fluorimetry and Western blotting using intact saphenous arteries and arterial pressure measurements under urethane anaesthesia. RESULTS: In 10- to 12-week-old rats, methoxamine (MX) produced vasoconstriction associated with an increase in [Ca2+ ]i and Ca2+ -sensitivity. In contrast, in 1-week-old rats these contractions were accompanied only by an increase in Ca2+ -sensitivity. All MX-induced effects were reduced by the Rho-kinase inhibitor Y-27632; this reduction was complete only in 1-week-old rats. The Rho-kinase specific site Thr855 on MYPT1 was increasingly phosphorylated by MX in vessels of 1-week-old, but not 10- to 12-week-old rats; this effect was also inhibited completely by Y-27632. The Rho-kinase inhibitor fasudil in a dose not affecting the pressor response to MX in 4- to 5-week-old rats reduced it considerably in 1-week-old rats. CONCLUSION: Our results suggest that the higher Ca2+ -sensitivity of arterial contraction in 1-week-old compared to 10- to 12-week-old rats is due to a greater Rho-kinase activity. Constitutively active Rho-kinase contributes to MX-induced contraction in 10- to 12-week-old rats. In 1-week-old rats, additional Rho-kinase activation is involved. This remodelling of the Rho-kinase pathway is associated with its increased contribution to adrenergic arterial pressure responses.

Quercetin relaxes rat tail main artery partly via a PKG-mediated stimulation of KCa 1.1 channels.

AIM: Protein kinases, activated by vasodilator substances, affect vascular function by regulating large conductance Ca(2+) -activated K(+) (KCa 1.1) channels. Thus, the aim of the present investigation was to address the hypothesis that quercetin-induced vasorelaxation is caused by a PKG-mediated stimulation of KCa 1.1 currents. METHODS: Single freshly isolated myocytes and endothelium-denuded rings of the rat tail main artery were employed for electrophysiological and contractility measurements respectively. RESULTS: Quercetin relaxed vessels and increased KCa 1.1 currents in a concentration-dependent manner: both effects were antagonized by the specific KCa 1.1 channel blocker iberiotoxin. Stimulation of KCa 1.1 currents was fully reversible upon drug washout, markedly reduced by Rp-8-Br-PET-cGMPs, a PKG-inhibitor, but not affected by catalase. Quercetin shifted by 34.3 mV the voltage dependence of KCa 1.1 channel activation towards more negative membrane potentials without affecting its slope. Under conditions of tight functional coupling between sarcoplasmic reticulum Ca(2+) release sites and KCa 1.1 channels, quercetin decreased both the frequency and the amplitude of KCa 1.1 transient currents in a ryanodine-like manner. CONCLUSION: The natural flavonoid quercetin relaxes the rat tail main artery partly via a PKG-mediated stimulation of smooth muscle KC a 1.1 channels.

[Organ specificity in nitric oxide storage in the blood vessel walls during adaptation to hypoxia].

Addition of N-acetylcysteine induced relaxation of the coronary and basilar arteries thus indicating some basilar NO-stores in these vessels. The maximum capacity of the NO-stores was similar in the coronary and the basilar arteries. Following adaptation to hypoxia, however, the depot was much greater in the coronary artery wall. This seems to be connected with different degree of participation of the NO-dependent vasodiatation in implementation of the adaptive response to hypoxia in coronary and cerebral vascular systems.

[Comparative analysis of vasomotor responses and sympathetic innervation of feed arteries from locomotor and respiratory muscles in a rat].

The characteristics of feeding arteries of diaphragm and medial gastrocnemius (with a diameter of 200-250 micron) were studied. The registration of the mechanical activity of ring preparations under isometric conditions revealed that diaphragm arteries, like arteries of other muscles with a high content of slow muscle fibers, are highly sensitive to adrenoceptor agonists and acetylcholine. The differences in endothelium-dependent relaxation between diaphragm and gastrocnemius arteries are preserved in the presence of L-NAME and diclofenac. Responses to serotonin in diaphragm and gastrocnemius arteries are similar. At the same time, the high density of innervation is characteristic of diaphragm artery only, while in other slow muscles it is low. The density of adrenergic fibers plexus in the diaphragm artery is much higher than in the gastrocnemius artery. The results suggest that the properties of small arteries of diaphragm are determined not only by the oxidative capacity of diaphragm muscle fibers but also by the belonging of the diaphragm to respiratory musculature.

[The contribution of protein kinase C and Rho-kinase to the control of the receptor-dependent artery contraction decreases with age independently of sympathetic innervation].

The age-related dynamics of the activity of signalling pathways coupled to alpha1-adrenergic receptors and their dependence on the sympathetic innervation of arterial smooth muscle have been studied. The effects of the protein kinase C inhibitor (GF109203X, 10(-6) M) and the Rho-kinase inhibitor (Y27632, 10(-5) M) on the isometric contraction of the rat saphenous artery, induced by the alpha1-adrenoceptor agonist methoxamine, were examined. It was shown that the sensitivity to methoxamine of arteries from 2-week-old rats that are partially innervated was reduced as compared to adults, but the effects of both inhibitors were more prominent. The denervation induced by the excision of sympathetic ganglia increased the arterial sensitivity to methoxamine but was not accompanied by changes in sensitivity to the inhibitors. Therefore, the postnatal development of the arterial smooth muscle is characterized by a decrease in the contribution of protein kinase C and Rho-kinase to the regulation of contraction; however, these changes do not correlate with changes in the sensitivity of arteries to methoxamine and development of sympathetic innervation.

[Elevation of the vascular smooth muscle sensitivity to effects of constrictors after denervation and under decreased blood pressure].

Changes in contractile activity of saphenous artery in normotensive rats and in rats with regional hypotension have been investigated. The abdominal aorta was partially occluded in Wistar rats distally to the renal arteries. Four weeks later, a 5-7-mm segment of the femoral nerve in one hindlimb was resected to denervate the saphenous artery. After two weeks, the isometric contraction of innervated and denervated saphenous artery segments was studied. In normotensive rats, the denervation augmented vessel sensitivity to noradrenaline, phenylephrine, serotonin, and KCl (in the presence of phentolamine). Chronic hypotension also augmented vessel sensitivity to constrictor agonists, whereas denervation did not result in further increase of sensitivity. In glyoxilic acid-stained preparations obtained from hypotensive rats, a reduced intensity of fluorescence of adrenergic fibers was observed. It was assumed that the higher sensitivity of vascular smooth muscle in hypotensive rats is due to functional disturbances of sympathetic innervation.

[The growth stimulation of a cell culture of dissociated rat embryonic spinal cord under the influence of the opioid peptide dalargin and the opioid receptor blocker naloxone]. / Stimuliatsiia rosta kul'tury kletok dissotsiirovannogo spinnogo mozga émbrionov krys pod vliianiem opioidnogo peptida dalargina i blokatora opioidnykh retseptorov naloksona.

Effect of synthetic analog of [Leu]-enkephalin dalargin and naloxone as a opioid blocker on fetal rat spinal cord cells was studied. It was found that the aggregation place during dalargin action had increased on average by 1.85 compared to the control, during naloxone action had increased on average by 1.78 compared to the control. The fact that brain cells reactions on opioid and on naloxone are the same and naloxone-peptide mixture is more active by 2.5 times compared to control suggests that these agents effect the cells with the help of receptors of different types, thus these agents are growth factors for nervous tissue, they increase survival and adhesion of the cells of CNS.

[The effect of opioid peptides on the size of the cellular aggregates in a cell culture of rat embryonic spinal cord]. / Vliianie opioidnykh peptidov na razmery kletochnykh agregatov v kul'ture kletok spinnogo mozga émbrionov krys.

Effect of opioid peptide Leu-enkephalin (Leu-enk) and synthetic analogue of Leu-enk on fetal rat spinal cord cells in culture was studied. Activity of peptides was measured in dissociated culture on aggregation assay. The aggregated place was calculated on days 3 and 6 of culturing, because this parameter indicates the cell adhesion and neuronal and glial survival. The aggregation place was shown to increase due to peptide action: with Leu-enk and dalargin by 1.6 and 1.7, resp., compared to the control; both calculations being made on days 3 and 6 of culturing. The results of the analysis of opioid peptide activity during culturing of dissociated cells of fetal rat spinal cord demonstrate that the peptides changed the adhesion of cells and increased their survival. The results of the present and our earlier experiments suggest that the opioid peptides may be growth factors for CNS cells and may play a role in development and regeneration of the nervous tissue.

[The growth-stimulating action of the preparation Balis-2 on sympathetic ganglia in culture]. / Roststimuliruiushchee deistvie preparata baliz-2 na simpaticheskie ganglii v kul'ture.

The effect of Balis-2 drug on the growth and differentiation of nervous tissue was studied on organotypic culture of the sympathetic ganglia. It was established that this agent is able to stimulate fiber outgrowth from explant and increase mean value of the maximal magnitude of the zone of the growth by concentration 0.001% and 0.0001%. It was found that Balis-2 drug is able to increase the intensity of reaction by 1.8-2 times. It is suggested that Balis-2 drug can be used as a new neurotropic agent.

[Effect of the preparation baliz-2 on the growth of cultures of sympathetic ganglia from various strains of rats]. / Vliianie preparata baliz-2 na rost kul'tur simpaticheskikh gangliev krys razlichnykh linii.

The effect of the baliz-2 drug (0.01%, 0.001%, 0.0001%) on the growth and differentiation of nervous tissue was studied on organotypic cultures of the sympathetic ganglia of newborn Wistar, Wag, August rats. The growth zone of explants in living cultures and histochemical stained preparations were analyzed for catecholamine-containing neurites. It has been established that control value of growth intensity for August ganglia is 2.1 2.0 times lower than for Wistar and Wag. The baliz-2 drug at concentration 0.001% and 0.0001% is able to stimulate fibre outgrowth from the explant. Mean values of the reaction are different: they are maximal for August rats (2.3-2.6 times higher than the control), while for Wistar and Wag rats they are 1.8-2.0 times higher than the control. These results provide evidence that the baliz-2 drug is a neurotrophic agent for sympathetic ganglia in culture. The results are discussed in terms of the role of the sympathetic-adrenal system in the nervous tissue regeneration.

[Activating effect of substance P on nerve tissue in culture]. / Aktiviruiushchee deistvie veshchestva P na nervnuiu tkan' v kulture.

The effect of substance P on organotypic culture of rat sympathetic ganglia and spinal cord was studied. Substance P was able to stimulate the fibre outgrowth from explant, to increase the numbers of glial and fibroblasttypic cells in ganglia growth zone. The effect was observed in the range from 10(-5) to 10(-12) M (for sympathetic ganglia) and from 10(-5) to 10(-14) M (for spinal cord culture). It is suggested that substance P can be used as a nonspecific growth factor for peripheral and central nervous tissue.

[Features of the action of opioid peptides and naloxone on the tissues of the central and peripheral nervous system during cultivation]. / Osobennosti deistviia opioidnykh peptidov i naloksona na tkan' tsentral'noi i perifericheskoi nervnoi sistemy v usloviiakh kul'tivirovaniia.

The effect of opioid peptides (leu-enkephalin, a synthetic analog of leu-enkephalin) and naloxone on the organotypic culture of rat spinal cord and dorsal root ganglia was studied. The explants' outgrowth and dorsal root ganglion neuron survival were studied. Peptides (10(-9) M-10(-10)M) and naloxone (10(-5) M-10(-6) M) were found to be able to stimulate fibre outgrowth from the explant, to increase the numbers of glia and fibroblast type cells in the ganglion growth zone. It is suggested that opioid peptides can be used as a source of nonspecific growth factor for nervous tissue of peripheral and central nerve system.

[Action of opioid peptides on nerve tissue growth and regeneration processes in the rat]. / Deistvie opioidnykh peptidov na protsessy rosta i regeneratsii nervnoi tkani krysy.

Using organotypic cultures of the sympathetic ganglia and spinal cord from rats, studies have been made of the effect of opioid peptides on the development of the nervous tissue. It was found that endogenous opioid peptides (leu- and met-enkephalins, beta-endorphin) within the concentrations investigated (10(-9)-10(-10) M), stimulate the growth of neurites, affect the rate of migration and proliferation of the glial and fibroblast-like cells. The effect was observed at the 2nd--5th days of cultivation, depending on the object investigated. Naloxone, a blockator of the opiate receptors, does not abolish the stimulating effect of the opioid peptides. Using clonal line of fibroblast-like cells L6, it was shown that leu-enkephalin decreases the sensitivity to contact inhibition of growth. On the basis of the data obtained, it is suggested that endogenous opioid peptides act as non-specific factors of growth regulation in the development and regeneration of the nervous tissue. Taking into account the role of endorphins in the activity of noci-antinociceptive system possible significance of these compounds in post-injury reparation is discussed.