Modulation of the vascular function by cannabinoids, calcitonin gene related peptide and connexin hemichannels

Blood vessel diameter and blood flow are tightly controlled by electrical signals and calcium ion (Ca2+) signals in smooth muscle cells (SMCs) and endothelial cells (ECs) in which selective and non-selective ion channels play a crucial role. In this doctoral thesis we characterized the targets of cannabinoid- and calcitonin gene related peptide (CGRP)-triggered vasodilation and the role of connexin channels in the modulation of blood vessel tone. Additionally, we investigated connexin channels as a putative target to protect blood vessels against cell death associated with stressful conditions like cryopreservation. Blood vessel tonus can be modulated by several factors, such as endothelial factors or factors released from perivascular nerves, thereby promoting vasodilation or vasoconstriction. Cannabinoids have been used for centuries for their psychoactive properties, but they have also a profound influence on the cardiovascular system, leading to vasodilation. Vasorelaxation by cannabinoids can be mediated by stimulation of cannabinoid receptors. Others pointed a role for vanilloid receptors on perivascular nerves and a subsequent release of the neuropeptide CGRP. This peptide is one of the most potent vasodilatory substances and can act via an endothelium-dependent or -independent process. In order to find out if a direct effect of the cannabinoid methanandamide was present in small mesenteric rat arteries, we performed experiments on acutely isolated SMCs. Our findings show that the cannabinoid methanandamide fails to increase the membrane potassium (K+) currents and fails to hyperpolarize the membrane potential. Moreover, the neuropeptide CGRP is acting specifically and directly on these cells by increasing the large-conductance calcium-dependent potassium (BKCa) channel activity in a receptor-, cyclic adenosine monophosphate- (cAMP), and protein kinase A- (PKA) dependent way and hyperpolarizes the membrane potential of these cells. This is consistent with earlier reports suggesting that methanandamide relaxes and hyperpolarizes intact rat mesenteric arteries by releasing the neuropeptide CGRP from perivascular nerves. Next to electrical changes, vessel diameter and blood flow are also controlled by Ca2+ signals and communicative pathways between vascular cells in which gap junctions (GJs) play a crucial role. GJs are dodecameric channels composed of connexin proteins that directly connect the cytoplasm of cells. These intercellular channels are composed of two hexameric 10 hemichannels (HCs) that may also be junctional as non-junctional HCs. Non-junctional (unapposed) HCs are present in the plasma membrane normally closed but may open by various messengers and conditions thereby forming a pore that allows passage of ions and messengers like ATP or other substances with a molecular weight below 1.5 kDa. HC opening allows Ca2+ entry into the cells but opening is also controlled by the intracellular (cytoplasmic) Ca2+ concentration ([Ca2+]i). Therefore, these channels are likely to contribute to Ca2+ oscillations which are repetitive [Ca2+]i changes. We investigated the role of HCs in Ca2+ oscillations by interfering with HC function via the application of peptides that are identical to connexin sequences on the connexin protein. We found that in small mesenteric artery fragments isolated from rat, norepinephrine-induced Ca2+ oscillations in SMCs were inhibited by the connexin mimetic peptide Gap27, by the TAT-L2 peptide and by the TAT-CT9 peptide. Gap27 inhibits HCs first and with some delay also GJs; it targets multiple connexins because it mimics well-conserved extracellular domains of the connexin protein. By contrast, TAT-L2 peptide has specificity at two levels: it only inhibits HCs but not GJs and it has specificity for Cx43 and not for other connexins. The latter is related to the fact that this peptide is identical to a part of the intracellularly located non-conserved domain. TAT-CT9 also has specificity for Cx43 but this peptide prevents the closure of HCs at high micromolar [Ca2+]i. In addition to allowing Ca2+ entry, open HCs can also allow the escape of ATP from the cell. We found that interfering with purinergic signaling, by promoting the degradation of ATP or by inhibiting purinergic receptors, norepinephrine-induced Ca2+ oscillations were also inhibited. Taken together, these findings suggest that ATP released by SMCs via connexin hemichannels (CxHCs) with subsequent activation of purinergic communication is involved in controlling vessel responses to norepinephrine. Tension measurements further confirmed the role of CxHCs in vascular smooth muscle tension since the contractility was also inhibited by Gap27, by TAT-L2 and by purinergic receptor antagonists. These data indicate that interfering with CxHCs offers a novel approach towards modulating blood vessel reactivity. GJs and CxHCs are important for vascular function, but they also play a role in cell death processes. GJs allow the passage of cell death messengers thereby contributing to the propagation of cell death known as bystander cell death. Unapposed CxHCs may promote cell death by uncontrolled opening and thereby contribute to the entrance or escape from 11 the cell of ions and small metabolites. Inhibiting connexin channels may thus lead to improved cell viability after exposure to stressful conditions like exposure to freezing conditions in the context of cryopreservation of cells and tissues. Cryopreserved blood vessels are being increasingly employed as grafts in vascular reconstruction procedures. However, the massive cell death provoked by the methods of cryopreservation used at most vascular banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vascular allografts. We investigated whether blocking connexin channels could prevent cell death after cryopreservation. We report that Gap27 significantly reduced cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. Veins had a better conserved intimal layer compared to arteries and had less endothelial cell death. This implies that veins are better suited for vessel replacement in patients with vascular diseases and a high risk of thrombosis because of the anti-thrombogenic effect of an intact endothelium. Most notably, blocking GJs and HCs with connexin mimetic peptides like Gap27 appears to be very efficient to prevent cell death in complex tissues like blood vessels undergoing cryopreservation. It is important to note here that blocking GJs and HCs is (in most cases) are not indicated in vivo because GJs are necessary for normal physiological function. However, inhibition of GJs and HCs is perfectly possible in tissues and organs isolated ex vivo for subsequent cold or cryo storage. Such procedure may well be indicated in the context of heart, lung and kidney transplantation and cryopreservation/vitrification of various cells and tissues. In conclusion, this work has improved our understanding of the vascular action of cannabinoids and CGRP, and has brought up connexins and their channels as interesting new targets to modulate blood vessel function and to protect vascular cell viability after cryopreservation

Targeting connexins with Gap27 during cold storage of the human donor uterus protects against cell death

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4C) or warm (37C) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority

Influence of methanandamide and CGRP on potassium currents in smooth muscle cells of small mesenteric arteries

Cannabinoids have potent vasodilatory actions in a variety of vascular preparations. Their mechanism of action, however, is complex. Apart from acting on vascular smooth muscle or endothelial cannabinoid receptors, several studies point to the activation of type 1 vanilloid (TRPV1) receptors on primary afferent perivascular nerves, stimulating the release of calcitonin gene-related peptide (CGRP). In the present study, the direct influence of the cannabinoid methanandamide and the neuropeptide CGRP on the membrane potassium ion (K+) currents of rat mesenteric myocytes was explored. Methanandamide (10 mu M) decreased outward K+ currents, an effect similar to that observed in smooth muscle cells from the rat aorta. Conversely, CGRP (10 nM) significantly increased whole-cell K+ currents and this effect was abolished by preexposure to tetraethylammonium chloride (1 mM) or iberiotoxin (100 nM), inhibitors of large-conductance calcium-dependent K (BKCa) channels but not by glibenclamide (10 mu M), an inhibitor of ATP-dependent K channels. In the presence of the CGRP receptor antagonist CGRP(8-37) (100 nM), the adenylyl cyclase inhibitor SQ22536 (100 mu M), or the protein kinase A inhibitor Rp-cAMPS (10 mu M), CGRP had no effect. These findings show that methanandamide does not increase membrane K+ currents in smooth muscle cells of small mesenteric arteries, supporting an indirect mechanism for the reported hyperpolarizing influence in this vessel. Moreover, CGRP acts directly on these smooth muscle cells by increasing BKCa channel activity in a CGRP receptor and cyclic adenosine monophosphate-dependent way. Collectively, these data indicate that methanandamide relaxes and hyperpolarizes intact mesenteric vessels by releasing CGRP from perivascular nerves