Fig. 1
Receptor desensitization mechanism of vascular hyporesponsiveness. The signal transductions for receptor desensitization include three pathways. One is the internalization of surface receptors, the second pathway is receptor transcription regulation through JAK2-STAT3 and NF-kB related pathways. Both of these two pathways might down-regulate the receptor amount. The third pathway is receptor uncoupling and receptor affinity regulation through G-proteins. EOP endogenous opioid peptide; JAK2 janus activating kinase 2; STAT3 signal transducer and activator of transcription 3; GPCR G protein coupled receptor; AR adrenergic receptor; AC adenylyl cyclase
Receptor affinity decrease is another important mechanism for receptor desensitization. It is often seen before the decrease of receptor number on the cell membrane surface. Previous studies showed that the receptor affinity of β-AR is generally declined during endotoxic and hemorrhagic shock but the affinity of α-AR remains constant [42, 43]. Decease of receptor affinity may also result in receptor uncoupling, and thereby decrease the activity of adenylyl cyclase (AC). Therefore, the coupling obstacles between adrenergic receptors and adenylyl cyclase may be the most important factor for receptor desensitization.
Membrane hyperpolarization mechanism Studies showed after hemorrhagic and burn shock , vascular smooth muscle cell (VSMC) would appear membrane hyperpolarization. Membrane hyperpolarization is a crucial mechanism to vascular hyporesponsiveness after shock. VSMC membrane hyperpolarization mainly involves in two kinds of potassium channels including ATP-dependent K+ channel (KATP) and large conductance Ca2+-activated K+ (BKCa) channel. In physiological conditions, cytoplasm ATPs are in mmol–magnitude, which is enough to completely close the KATP channels on cell membrane [44]. While in shock condition, the disorders of the cell oxidative metabolism or the huge reduction of ATP would cause the open of KATP channels on cell membrane [45, 46]. The over-opened KATP channels in VSMC would result in membrane hyperpolarization of VSMC. This process would inhibit the potential dependent calcium channel, decrease the Ca2+ inflow, and finally result in vascular hyporesponsiveness . The inducing factors for over opening of KATP channels include ATP decrease, nitric oxide (NO) production, and so on.
Although KATP channel plays an important effect in VSMC membrane hyperpolarization and vascular hypo-responsiveness following shock, the density of KATP channel in VSMC is only one channel per 10 µm2. However, BKCa channel, not only broadly distributes on VSMCs (its density in VSMC is 1–4 channels/µm2), but also plays an important regulatory role in vascular reactivity [47]. BKCa channel consists of α-subunit and accessory β-subunit, which co-affect the characteristics of the physiology and pathophysiology of BKCa channel [48]. Studies showed that Ca2+ sparks are the physiological activators of BKCa Channels. A single Ca2+ spark may cause the open of its surrounding BKCa channel and K+ outflow, which forms spontaneous transient outward current (STOC) [47]. This process may induce membrane hyperpolarization. In turn, over-opened BKCa channels cause the decrease of external calcium influx and finally results in the vascular hyporesponsiveness (Fig. 2).
Fig. 2
Membrane hyperpolarization mechanism of vascular hyporesponsiveness. The membrane hyperpolarization of vascular smooth cell after shock is mainly involved in two channels- KATP channel and BKCa channel. The membrane hyperpolarization of vascular smooth cell may inhibit the open of VOC, and via which results in the decrease of intracellular [Ca2+] and vascular hyporesponsiveness. K ATP channel ATP-dependent K+ channel; BKCa channel large conductance Ca+– activated K+ channel; VOC voltage-dependent calcium channel; CO carbon Oxide; EOP endogenous opioid peptide; PTK protein tyrosine kinase; PTP protein tyrosine phosphatase
3.2 Calcium Desensitization Mechanism
An interesting phenomenon should be concerned is that restoration of adrenergic receptor, K+ and Ca2+ channels’ functions cannot return the vascular reactivity to normal level, which suggests that there are other ways to regulate the vascular reactivity following shock. The key event of receptor desensitization and membrane hyperpolarization mechanism responsible for vascular hyporesponsiveness is the decrease of intracellular [Ca2+]. While at late stage of shock or in severe shock , the intracellular calcium in VSMCs is over loaded, but the vascular hyporesponsiveness still exists [5]. This suggests there may be other mechanisms that participate in the occurrence of vascular hyporesponsiveness after shock. Based on the basic theory that the contractile force of VSMC is dependent on the ratio of force and calcium, our research group raised the calcium desensitization hypothesis for vascular hyporesponsiveness. With in vivo and in vitro, and animal and cell experiments, we found that following shock, VSMC indeed exists calcium desensitization. Calcium desensitization played very important effect in vascular hyporesponsiveness [49]. That is to say the occurrence of vascular hyporesponsiveness have calcium desensitization mechanism. Our further studies found that Rho kinase and PKC pathway are the key pathways in calcium sensibility regulation of vascular reactivity following shock (Fig. 3).
Fig. 3
Calcium desensitization mechanism of vascular hyporesponsiveness. RhoA regulates vascular reactivity mainly through activation of Rho kinase and inhibition of Rac1 while Rac1 regulates vascular reactivity mainly through inhibition of RhoA and activation of PAK. Rho kinase may be through three pathways to exert effect: directly activate MLC20, inhibit MLCP and via CPI-17. ZIPK and ILK are the important downstream molecules of PKC. ZIPK may directly bind with CPI-17. ERK and p38MAPK play effect mainly through ILK and ZIPK or CPI-17. A1AR and A3AR may enhance the activity of PKCε and RhoA while A2aAR and A2bAR may inhibit the activity of RhoA and PKCε. RhoA Ras homolog gene family member A; Rac Ras related C3 botulinum toxin substrate; PAK P21-activated kinase; MLCP myosin light chain phosphatase; MLCK Myosin light chain kinase; MLC 20 20-kDa myosin light chain; PKC protein kinase C; MAPK Mitogen-activated protein kinase; ERK extracellular signal-regulated kinase; CPI-17 protein kinase C-dependent phosphatase inhibitor of 17 kDa; ILK integrin linked kinase; ZIPK Zipper- interacting Protein Kinase; AR adenosine receptor
Role of Rho A-Rho kinase pathway in the regulation of calcium sensitivity Rho kinase, a Ser/Thr protein kinase, is a GTP-Rho binding protein. Previous studies showed that Rho kinase participates in the regulation of many biological process of cells, such as cell proliferation, cell differentiation and migration of tumor cells, the migration and invasion of trophoblast cells, etc. [50]. Our research group and Schmitz et al. found that Rho kinase plays critical role in the regulation of vascular reactivity and calcium sensitization following hemorrhagic shock [4, 50].
Basic research showed the calcium sensitivity regulation of VSMC depends on the phosphorylation and dephosphorylation of myosin light chain (MLC), which is regulated by myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), respectively [51, 52]. Studies showed that there are three ways for Rho kinase regulating the calcium sensitivity of VSMC (Fig. 3): (1) Rho kinase phosphorylate MLC20 directly. The strength of Rho kinase phosphorylating MLC20 is about one third of MLCK. So this way is not the main way that Rho kinase regulates calcium sensitivity after hemorrhagic shock. (2) The main way of Rho kinase regulating calcium sensitivity is that Rho kinase phosphorylates myosin-binding subunits (MBS) of MLCP at Thr2695, Thr2850 and Ser2854, and via which inhibits the activity of MLCP and increases the phosphorylation level of MLC20 [53]. (3) Rho kinase can also activates CPI-17 via phosphorylation of the Thr238 site of CPI-17. The activated CPI-17 enhances the phosphorylation of MLC20 through inhibiting the MLCP.
Role of PKC pathway in the regulation of calcium sensibility regulation Protein kinase C (PKC), a Ser/Thr protein kinase, plays a critical role in cell adaptability to extracellular environment. PKC is also involved in varieties of physiologic functions including cell proliferation, differentiation and migration, cytoskeletal structure, and apoptosis [54, 55]. PKC is a big family consisting of at least 12 isoforms, and the main isoforms distributed in the vascular system are PKC α, ε, δ and ξ. Basic research showed that the various isoforms of PKC, especially α and ε isoforms, may be activated through subcellular redistribution and transfer from cytoplasm to membrane, and then trigger a series of cascade reactions that ultimately interacts with the contractile myofilaments and leads to VSMC contraction [56].
Many studies showed that PKC participated in the regulation of vascular reactivity and calcium sensitivity following shock. Our previous study found that PKC agonist, phorbol-12- myristate-13-acetate (PMA), could improve and stabilize the hemodynamic parameters and play beneficial effect for hemorrhagic shock in rats through improving the vascular reactivity and calcium sensitivity [8, 18]. There are several mechanisms that PKC regulates the vascular reactivity and calcium sensitivity [57–59]: (1) The study of Woodsome et al. showed that PKC may phosphorylate CPI-17, and then inhibits the MLCP activity, via which increases the MLC20 phosphorylation and calcium sensitivity of VSMC [56]. (2) Our recent studies showed that the inhibitory effect of PKC on MLCP is not only related to CPI-17 but also related to integrin-linked kinase (ILK) [60, 61] and zipper-interacting protein kinase (ZIPK) [61]. Our results showed that ZIPK and ILK may be the direct downstream molecules of PKC α and ε, in which CPI-17 may play an indirect modulating role on MLCP. Our very recent study found that Rho kinase is the downstream molecule of ILK and ZIPK, and the upstream molecule of CPI-17 (Fig. 3) [25].
Role of MAPK pathway in the regulation of calcium sensibility Mitogen-activated protein kinases (MAPKs) belong to a family of serine/threonine protein kinases, which include extracellular-signal regulated kinase (ERK), jun NH2 -terminal kinase (JNK), and p38 MAPK in mammals. It was reported that MAPKs mediated the fundamental biological process to external signals, such as cytokines and inflammatory mediators [5]. Previous studies showed that MAPK had a critical role in regulating cell differentiation, proliferation and cell death [62, 63]. Our recent study [5] investigated the potential role of MAPK in the regulation of vascular reactivity and calcium sensitivity after hemorrhagic shock and interestingly found that the activity of ERK and p38MAPK were positively correlated with the change of vascular reactivity after hemorrhagic shock [4]. In SMAs, the activity of ERK and p38MAPK was significantly increased at early stage of shock (0.5 h) and decreased after prolonged shock (2 h), the inhibitors of ERK and p38MAPK decreased the vascular reactivity and calcium sensitivity following shock. This finding suggests that MAPK pathway participates in the regulation of vascular reactivity and calcium sensitivity following shock.
Role of adenosine and its receptor in the regulation of calcium sensibility Adenosine is one of the most important endogenous modulator released excessively after severe trauma and ischemia or hypoxia in tissue. It has been demonstrated that adenosine mainly produces the marked effect through adenosine receptor in VSMC. There are four types of adenosine receptor (AR) reported in VSMCs, including A1AR, A2aAR, A2bAR and A3AR. Adenosines which combine with specific AR may cause vasoconstriction (A1AR) or vasodilatation (A2aAR, A2bAR). The study of Srinivas et al. reported that exogenous adenosine may reduce the phosphorylation of MLC in bovine cornea epithelial cells [64]. However, the report of Lai et al. demonstrated that exogenous adenosine may induce MLC phosphorylation on VSMCs and increase its calcium sensitivity [65]. The results suggest that adenosine is closely correlated with vascular reactivity and calcium sensitivity .
Huang et al. [53] demonstrated that A1AR agonist (N6-cyclopentyladenosine, CPA, 10−5 mol/L) can induce renal artery constriction, which can be antagonized by Rho kinase inhibitor (Y-27632). This indicates that Rho kinase is correlated with A1AR in the regulation of vascular tone. The study of Tawfik et al. [66] showed that PKC inhibitor U-73122 could abolish the vasoconstriction induced by A1AR and A1AR agonist may enhance the activity of PKCε. This indicates that A1AR regulating vascular calcium sensitivity is also related to PKC pathway.
However, A2aAR and A2bAR can activate adenylate cyclase which causes the increase in cAMP concentration and PKA activation. The activated PKA may inhibit the activity of Rho kinase. This will induce MLC dephosphorylation and vascular smooth muscle dilation. Besides, the study of Gardner et al. showed that A2aAR may down-regulate the activity of PKC and A2aAR agonist CGS21680 may inactivate PKCε [67].
Our research group and Abbracchio et al. found that A3AR is also involved in the modulation of vasoreactivity following shock and this regulation is closely related to Rho kinase pathway [68, 69]. Furthermore, Zhao et al. reported that the A3AR agonist IB-MECA could up-regulate PKCδ activity and A3AR antagonist MRS-1191 could counteract this function [52], which suggests that A3AR regulating vascular reactivity and calcium sensitivity is related to PKCδ pathway.
4 The Approaches Towards Vascular Hyporesponsiveness Based on Calcium Desensitization Mechanisms
Based on the various vascular hypo-reactivity theories and its inducing factors, the therapeutic approaches may occur through blocking the related pathways.
4.1 Based on Receptor Desensitization and Membrane Hyperpolarization
Based on receptor desensitization Glucocorticoid (GC) may promote the catecholamine biosynthesis and potentiate the vasoconstriction effect of vasopressin (AVP), Angiotensin II and endothelin (ET) by increasing the sensitivity of their receptors [70–72]. In addition, studies showed cortisol has significant inhibitory effect on pro-inflammatory mediators, such as TNF-α and IL-1β, which are confirmed to be correlated with receptor desensitization [73]. A recent study showed that dexamethasone may rapidly reverse LPS induced hyporesponsiveness of VSMC to NE and this effect of dexamethasone is related to increasing the phosphorylation of MLC20 via increasing activity of the RhoA-Rho kinase pathways. These results demonstrated that glucocorticoid increasing the vascular reactivity is not only related to increasing the sensitivity of related receptors, but also related to increasing the calcium sensitivity of VSMC [57, 58].
There still remains controversy about the application of small doses of GC in patients with trauma or shock. The Surviving Sepsis Campaign Guidelines (2012) [59] suggest if hemodynamic stability can be achieved via adequate fluid resuscitation and vasopressor therapy, intravenous hydrocortisone is not allowed to use in adult septic shock patients. If this is not achievable, 200 mg of intravenous hydrocortisone per day is recommended. In addition, the guidelines also suggest septic shock patients if they should receive hydrocortisone do not use the ACTH stimulation test. When vasopressors are no longer required corticosteroids should be reduced or stopped. If no shock exists, do not use corticosteroids for the treatment of sepsis [59].
Based on membrane hyperpolarization Glybenclamide is a kind of KATP channel antagonist. Zhao et al. [74] found that application of glybenclamide combined with NaHCO3 could significantly increase the vascular reactivity in hemorrhagic rats. The blood pressures, arteriolar blood flow as well as the 24-h survival rate were also markedly increased after administration of glybenclamide, which indicates that glybenclamide combined with NaHCO3 is an effective regimen in the treatment of severe hemorrhagic shock. Studies showed NO generated OONO− with superoxide anion can induce the membrane hyperpolarization of VSMC though activation of KATP channels. Superoxide anion scavenger Tiron may block the production of OONO− in hemorrhagic shock rats, and inhibit the membrane hyperpolarization of VSMC and improve shock-induced vascular hyporesponsiveness [75].
As a hotspot in recent years, mitochondrial function has important value in the genesis of many diseases, such as cardiovascular diseases, Alzheimer disease, and Parkinson disease, and so on. Mitochondrial dysfunction also takes part in the occurrence of vascular hypo-responsiveness after shock. Zhao et al. found that Polydatin, a mitochondrial protector, can protect the mitochondria of vascular smooth muscle and restore the vascular reactivity in hemorrhagic shock rats [76, 77]. Our recent study found that cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition pore (MPTP), and 4-Phenylbutyrate (PBA), an anti-oxidation agent, could improve the vascular reactivity of traumatic hemorrhagic shock in rats via inhibition of oxidative stress and protecting the mitochondrial function [78, 79].
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