Mária Szekeres

Angiotensin II Induces Vascular Endocannabinoid Release, Which Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors

Background: In expression systems diacylglycerol (DAG) produced during AT1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol. Results: Inhibition of CB1 receptors and DAG lipase augmented angiotensin II-induced vasoconstriction in resistance arteries. Conclusion: Angiotensin II-induced vasoconstriction is attenuated via 2-arachidonoylglycerol release and consequent CB1 receptor activation. Significance: This is the first demonstration that angiotensin II-induced endocannabinoid release can modulate vasoconstriction. In the vascular system angiotensin II (Ang II) causes vasoconstriction via the activation of type 1 angiotensin receptors. Earlier reports have shown that in cellular expression systems diacylglycerol produced during type 1 angiotensin receptor signaling can be converted to 2-arachidonoylglycerol, an important endocannabinoid. Because activation of CB1 cannabinoid receptors (CB1R) induces vasodilation and reduces blood pressure, we have tested the hypothesis that Ang II-induced 2-arachidonoylglycerol release can modulate its vasoconstrictor action in vascular tissue. Rat and mouse skeletal muscle arterioles and mouse saphenous arteries were isolated, pressurized, and subjected to microangiometry. Vascular expression of CB1R was demonstrated using Western blot and RT-PCR. In accordance with the functional relevance of these receptors WIN55212, a CB1R agonist, caused vasodilation, which was absent in CB1R knock-out mice. Inhibition of CB1Rs using O2050, a neutral antagonist, enhanced the vasoconstrictor effect of Ang II in wild type but not in CB1R knock-out mice. Inverse agonists of CB1R (SR141716 and AM251) and inhibition of diacylglycerol lipase using tetrahydrolipstatin also augmented the Ang II-induced vasoconstriction, suggesting that endocannabinoid release modulates this process via CB1R activation. This effect was independent of nitric-oxide synthase activity and endothelial function. These data demonstrate that Ang II stimulates vascular endocannabinoid formation, which attenuates its vasoconstrictor effect, suggesting that endocannabinoid release from the vascular wall and CB1R activation reduces the vasoconstrictor and hypertensive effects of Ang II.

Nitric Oxide and Prostaglandins Modulate Pressure-Induced Myogenic Responses of Intramural Coronary Arterioles

The myogenic response, an active constriction and dilation of vessels to changes in intravascular pressure, can play an important role in the regulation of coronary blood flow. The characteristics of the myogenic response and its modulation by endothelium-derived factors are organ and location specific and have not been studied extensively in intramural coronary arterioles. Thus, distal intramural branches (∼100 and ∼170 &mgr;m active and passive diameter, respectively) of the left anterior descending coronary artery of rats were isolated and cannulated. Step increases in intraluminal pressure from 0 to 40 mm Hg elicited increases in diameter, whereas further increases in pressure from 50 to 150 mm Hg resulted in constrictions. In control, the pressure-induced myogenic tone of coronary arterioles was 67.3 ± 2.7% of passive diameter (PD, obtained in Ca2+-free solution) at 60 mm Hg. Nω-nitro-L-arginine (L-NNA, 10−5 M), an inhibitor of nitric oxide synthase, reduced the initial arteriolar diameter (by 44.8 ± 5.1 &mgr;m at 2 mm Hg, P < 0.05) and significantly mitigated increases in diameter to lower pressures and constrictions to higher pressures (41.1 ± 5.6% of PD at 60 mm Hg). Administration of adenosine restored the initial diameter in the presence of l-NNA, but the increase in diameter to lower pressures and the decrease in diameter to higher pressures observed under control conditions remained greatly inhibited. Inhibition of prostaglandin synthesis, or PGH2/TxA2 receptors significantly reduced the constrictions to higher pressures as compared with control (indomethacin: from 57.9 ± 4.8% of PD to 67.0 ± 4.7% of PD at 150 mm Hg). Thus, because in isolated intramural coronary arterioles of rats a negative slope for the pressure-diameter curve develops only in the presence of nitric oxide and constrictor prostaglandins, they seem to be essential for the normal development of the myogenic response.

Pharmacologic Inhomogeneity Between the Reactivity of Intramural Coronary Arteries and Arterioles

We hypothesized that because of their size, anatomic location, and hemodynamic function, coronary arteries and arterioles would respond differently to vasoactive substances. Intramural arteries (281.7 ± 23.1 &mgr;m) and arterioles (77.3 ± 6.6 &mgr;m) of the left anterior descending coronary of rats were isolated and cannulated. Spontaneous tone was lower in arteries than in arterioles (81.1 ± 5.7 vs. 53.0 ± 3.9% of passive diameter, p < 0.05 at 60 mm Hg intraluminal pressure). Arterial tone was adjusted by the thromboxane receptor agonist U46619 (5 × 10 −8M) to reach an active tone close to that of arterioles. Bradykinin elicited dilations in both types of vessels. Acetylcholine (10 −6 –10 −5M) dilated arteries (by 42.6 ± 11.5 &mgr;m) but constricted arterioles (by 16.4 ± 9.3 &mgr;m). Sodium nitroprusside and adenosine elicited significantly greater dilations in arterioles than in arteries (by 7.9 and 11.9%, respectively, p < 0.05), whereas dilations to norepinephrine were similar. Inhibition of nitric oxide synthesis caused a significantly smaller constriction in arteries (10.2 ± 3.31%) than in arterioles (31.6 ± 6.9%) and completely blocked bradykinin-and acetylcholine-induced dilations, whereas it did not affect dilations to sodium nitroprusside, adenosine, and norepinephrine. Compared with arteries, arterioles have a greater spontaneous tone and enhanced nitric oxide modulation of basal tone and exhibit greater responsiveness to nitric oxide and adenosine. In addition, nitric oxide synthase is activated differently by pharmacologic stimuli in these segments. The qualitative and quantitative differences among vasoactive responses of coronary arteries and arterioles demonstrated in this study suggest segment-specific roles for endothelial and metabolic factors in regulation of coronary vascular resistance.

Angiotensin II-Induced Expression of Brain-Derived Neurotrophic Factor in Human and Rat Adrenocortical Cells

Angiotensin II (Ang II) is a major regulator of steroidogenesis in adrenocortical cells, and is also an effective inducer of cytokine and growth factor synthesis in several cell types. In microarray analysis of H295R human adrenocortical cells, the mRNA of brain-derived neurotrophic factor (BDNF), a neurotrophin widely expressed in the nervous system, was one of the most up-regulated genes by Ang II. The aim of the present study was the analysis of the Ang II-induced BDNF expression and BDNF-induced effects in adrenocortical cells. Real-time PCR studies have shown that BDNF is expressed in H295R and rat adrenal glomerulosa cells. In H295R cells, the kinetics of Ang II-induced BDNF expression was faster than that of aldosterone synthase (CYP11B2). Inhibition of calmodulin kinase by KN93 did not significantly affect the Ang II-induced stimulation of BDNF expression, suggesting that it occurs by a different mechanism from the CYP11B2-response. Ang II also caused candesartan-sensitive, type-1 Ang II receptor-mediated stimulation of BDNF gene expression in primary rat glomerulosa cells. In rat adrenal cortex, BDNF protein was localized to the subcapsular region. Ang II increased BDNF protein levels both in human and rat cells, and BDNF secretion of H295R cells. Ang II also increased type-1 Ang II receptor-mediated BDNF expression in vivo in furosemide-treated rats. In rat glomerulosa cells, BDNF induced tropomyosin-related kinase B receptor-mediated stimulation of EGR1 and TrkB expression. These data demonstrate that Ang II stimulates BDNF expression in human and rat adrenocortical cells, and BDNF may have a local regulatory function in adrenal glomerulosa cells.

Segmental Differences in Geometric, Elastic and Contractile Characteristics of Small Intramural Coronary Arteries of the Rat

The depedence of elastic and contractile properties on the caliber of small intramural coronary arteries was investigated in the rat in vitro. Different segments of the left anterior descending coronary artery branching system were prepared for microarteriography. The segments were cannulated at both ends, immersed in oxygenated normal Krebs Ringer (nKR) solution. Intraluminal pressure was changed at a rate of about 0.5 mm Hg/s between 0 and 150 mm Hg in repeated cycles. The outer diameter was continuously measured with microangiometry. Pressure-diameter curves were recorded after preconditioning pressure cycles in nKR, with PGF2α in the bath (7.5 × 10–6 M), and in maximal relaxation with papaverine (2.8 × 10–4 M). Biomechanical parameters were computed for vessels grouped according to their calibers (inner diameters: 50–150, 150–250, 250–350, >350 µm). Distensibility and contractility decreased with increasing caliber of the vessels, while the elastic modulus increased. Spontaneous tone was (at 100 mm Hg in mechanically preconditioned vessels) 18.8 ± 4.5, 8.4 ± 4.4, 9.7 ± 3.7 and 8.3 ± 3.8% in the four groups, respectively. PGF2α contraction was maximal around the 300-µm caliber. Our study is the first direct demonstration that intramural small coronary arteries exhibit characteristic variability in their elastic and contractile properties as a function of their caliber. Such differences may be important in segmentally specific control processes of the coronary microcirculation.

Mechanisms of angiotensin II-mediated regulation of aldosterone synthase expression in H295R human adrenocortical and rat adrenal glomerulosa cells

In adrenal zona glomerulosa cells angiotensin II (Ang II) is a key regulator of steroidogenesis. Our purpose was to compare the mechanisms of Ang II-induced changes in the expression level of early transcription factors NR4A1 (NGFIB) and NR4A2 (Nurr1) genes, and the CYP11B2 gene encoding aldosterone synthase in H295R human adrenocortical tumor cells and in primary rat adrenal glomerulosa cells. Real-time PCR studies have demonstrated that Ang II increased the expression levels of NR4A1 and NR4A2 in H295R cells within 1 h after stimulation, which persisted up to 6 h; whereas in rat adrenal glomerulosa cells the kinetics of the expression of these genes were more rapid and transient. Ang II also induced prolonged nuclear translocation of Nurr1 and NGFIB proteins in both cell types. Studies using MEK inhibitor (PD98059, 20 microM), protein kinase C inhibitor (BIM1, 3 microM) and calmodulin kinase (CAMK) inhibitor (KN93, 10 microM) revealed that in rat adrenal glomerulosa cells CAMK-mediated mechanisms play a predominant role in the regulation of CYP11B2. In accordance with earlier findings, in H295R cells MEK inhibition increased the expression of NR4A1, NR4A2 and CYP11B2 genes, however, it decreased the Ang II-induced gene expression levels, suggesting that ERK activation has a role in control of expression of these genes. No such mechanism was detected in rat glomerulosa cells. Sar1-Ile4-Ile8-AngII, which can cause G protein-independent ERK activation, also stimulated the expression of CYP11B2 in H295R cells. These data suggest that the previously reported CAMK-mediated stimulation of early transcription factors NGFIB and Nurr1 has a predominant role in Ang II-induced CYP11B2 activation in rat adrenal glomerulosa cells, whereas in H295R cells ERK activation and G protein-independent mechanisms also contribute to this process.

Endocannabinoid-mediated modulation of Gq/11 protein-coupled receptor signaling-induced vasoconstriction and hypertension

Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

Recovery and aging of serotonergic fibers after single and intermittent MDMA treatment in dark agouti rat

3,4‐Methylenedioxymethamphetamine (MDMA; ecstasy) is a popular party drug known to cause selective serotonergic damage. Here we examined the long‐term recovery and aging of serotonergic fibers and levels of brain‐derived neurotrophic factor (BDNF) after intermittent MDMA administration (15 mg kg−1 i.p. every 7th day for 4 weeks, MDMA ×4) and a single‐dose treatment (15 mg kg−1 i.p., MDMA ×1) in adolescent/young adult male Dark Agouti rats. After MDMA treatment, tryptophan hydroxylase‐immunoreactive fiber density decreased and then recovered in all brain regions. Recovery was more pronounced in the MDMA ×4 group compared with the MDMA ×1 group, but similar long‐term BDNF responses were found after both treatments. Twenty‐two months after treatment, there were fewer clusters of aberrant serotonergic fibers in the parietal cortex in the MDMA ×4 group compared with the MDMA ×1 group. There was no difference in the density of microglial cells or astrocytes in treated groups versus the control 22 months after the treatments. These results indicate that recovery of serotonergic fibers is faster after intermittent MDMA treatment than after single‐dose administration, and differences in BDNF levels per se are unlikely to account for this difference. Moreover, it seems that intermittent MDMA treatment attenuates the morphological signs of aging in serotonergic fibers. In addition, neither intermittent nor single‐dose MDMA exposition of young animals induces accelerated aging processes or neurodegeneration in senescence, as indicated by the unaltered densities of microglial cells and astrocytes in the treated groups compared with the control. J. Comp. Neurol. 519:2353–2378, 2011.

Angiotensin II-induced activation of central AT1 receptors exerts endocannabinoid-mediated gastroprotective effect in rats

The aim of the present study was to analyze whether angiotensin II via the endocannabinoid system can induce gastric mucosal protection, since transactivation of cannabinoid CB1 receptors by angiotensin AT1 receptor in CHO cells was described. Experimental ulcer was induced by acidified ethanol given orally in male Wistar rats, CB1(+/+) wild type and CB1(-/-) knockout mice. The compounds were administered intracerebroventricularly. It was found, that 1. Angiotensin II inhibited the ethanol-induced gastric lesions (11.9-191pmol); the effect of angiotensin II (191pmol) was inhibited by the CB1 receptor inverse agonist AM 251 (1.8nmol) and the inhibitor of diacylglycerol lipase (DAGL), tetrahydrolipstatin (0.2nmol). 2. Angiotensin II exerted gastroprotection in wild type, but not in CB1(-/-) mice. 3. The gastroprotective effect of angiotensin II (191pmol) was reduced by atropine (1mg/kg i.v.) and bilateral cervical vagotomy. In conclusion, stimulation of central angiotensin AT1 receptors via activation of cannabinoid CB1 receptors induces gastroprotection in a DAGL-dependent and vagus-mediated mechanism.

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca2+i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca2+i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca2+i levels of the heart. The fluorescence ratio technique was applied to estimate Ca2+i. The disappearance of mechanical activity of the heart preceded that of the Ca2+i transient in the first 2 min of ischemia. The slope of upstroke of the Ca2+i transient, reflecting Ca2+ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca2+ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca2+i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca2+i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca2+i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca2+i handling and sustained depression of mechanical performance.