pH-dependent modulation of voltage gating in connexin45 homotypic and connexin45/connexin43 heterotypic gap junctions.

Intracellular pH (pH(i)) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell-cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH(i) affects junctional conductance (g(j)) and g(j) dependence on transjunctional voltage (V(j)). To characterize V(j) gating, we fit the g(j)-V(j) relation using a stochastic four-state model containing one V(j)-sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V(j) across it. Using the model, we estimated gating parameters characterizing sensitivity to V(j) and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH(i) changes from 7.2 to approximately 8.0 shifted g(j)-V(j) dependence of Cx45 aHCs along the V(j) axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g(j) dependence on V(j). In contrast, acidification shifted g(j)-V(j) dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g(j) achieved under alkaline conditions showed that only approximately 4% of channels were functional. The acid dissociation constant (pK(a)) of g(j)-pH(i) dependence of Cx45/Cx45 GJs was approximately 7. The pK(a) of heterotypic Cx45/Cx43-EGFP GJs was lower, approximately 6.7, between the pK(a)s of Cx45 and Cx43-EGFP (approximately 6.5) homotypic GJs. In summary, pH(i) significantly modulates junctional conductance of Cx45 by affecting both V(j) gating and number of functional channels.

Transition to collateral flow after arterial occlusion predisposes to cerebral venous steal.

BACKGROUND AND PURPOSE: Stroke-related tissue pressure increase in the core and penumbra determines regional cerebral perfusion pressure (rCPP) defined as a difference between local inflow pressure and venous or tissue pressure, whichever is higher. We previously showed that venous pressure reduction below the pressure in the core causes blood flow diversion-cerebral venous steal. Now we investigated how transition to collateral circulation after complete arterial occlusion affects rCPP distribution. METHODS: We modified parallel Starling resistor model to simulate transition to collateral inflow after complete main stem occlusion. We decreased venous pressure from the arterial pressure to zero and investigated how arterial and venous pressure elevation augments rCPP. RESULTS: When core pressure exceeded venous, rCPP=inflow pressure in the core. Venous pressure decrease from arterial pressure to pressure in the core caused smaller inflow pressure to drop augmenting rCPP. Further drop of venous pressure decreased rCPP in the core but augmented rCPP in penumbra. After transition to collateral circulation, lowering venous pressure below pressure in the penumbra further decreased rCPP and collaterals themselves became a pathway for steal. Venous pressure level at which rCPP in the core becomes zero we termed the "point of no reflow." Transition from direct to collateral circulation resulted in decreased inflow pressure, decreased rCPP, and a shift of point of no reflow to higher venous loading values. Arterial pressure augmentation increased rCPP, but only after venous pressure exceeded point of no reflow. CONCLUSIONS: In the presence of tissue pressure gradients, transition to collateral flow predisposes to venous steal (collateral failure), which may be reversed by venous pressure augmentation.

Severe intraoperative bronchospasm treated with a vibrating-mesh nebulizer.

Bronchospasm and status asthmaticus are two of the most dreaded complications that a pediatric anesthesiologist may face. With the occurrence of severe bronchospasm and the inability to ventilate, children are particularly vulnerable to apnea and ensuing hypoxia because of their smaller airway size, smaller lung functional residual capacity, and higher oxygen consumption rates than adults. Nebulized medication delivery in intubated children is also more difficult because of smaller endotracheal tube internal diameters. This case demonstrates the potentially lifesaving use of a vibrating-mesh membrane nebulizer connected to the anesthesia circuit for treating bronchospasm.

Partial aortic occlusion and cerebral venous steal: venous effects of arterial manipulation in acute stroke.

Acute ischemic stroke therapy emphasizes early arterial clot lysis or removal. Partial aortic occlusion has recently emerged as an alternative hemodynamic approach to augment cerebral perfusion in acute ischemic stroke. The exact mechanism of cerebral flow augmentation with partial aortic occlusion remains unclear and may involve more than simple diversion of arterial blood flow from the lower body to cerebral collateral circulation. The cerebral venous steal hypothesis suggests that even a small increase in tissue pressure in the ischemic area will divert blood flow to surrounding regions with lesser tissue pressures. This may cause no-reflow (absence of flow after restoration of arterial patency) in the ischemic core and "luxury perfusion" in the surrounding regions. Such maldistribution may be reversed with increased venous pressure titrated to avoid changes in intracranial pressure. We propose that partial aortic occlusion enhances perfusion in the brain by offsetting cerebral venous steal. Partial aortic occlusion redistributes blood volume into the upper part of the body, manifested by an increase in central venous pressure. Increased venous pressure recruits the collapsed vascular network and, by eliminating cerebral venous steal, corrects perifocal perfusion maldistribution analogous to positive end-expiratory pressure recruitment of collapsed airways to decrease ventilation/perfusion mismatch in the lungs.

Cerebral venous steal equation for intracranial segmental perfusion pressure predicts and quantifies reversible intracranial to extracranial flow diversion.

Cerebral perfusion is determined by segmental perfusion pressure for the intracranial compartment (SPP), which is lower than cerebral perfusion pressure (CPP) because of extracranial stenosis. We used the Thevenin model of Starling resistors to represent the intra-extra-cranial compartments, with outflow pressures ICP and Pe, to express SPP = Pd-ICP = FFR*CPP-Ge(1 - FFR)(ICP-Pe). Here Pd is intracranial inflow pressure in the circle of Willis, ICP-intracranial pressure; FFR = Pd/Pa is fractional flow reserve (Pd scaled to the systemic pressure Pa), Ge-relative extracranial conductance. The second term (cerebral venous steal) decreases SPP when FFR < 1 and ICP > Pe. We verified the SPP equation in a bench of fluid flow through the collapsible tubes. We estimated Pd, measuring pressure in the intra-extracranial collateral (supraorbital artery) in a volunteer. To manipulate extracranial outflow pressure Pe, we inflated the infraorbital cuff, which led to the Pd increase and directional Doppler flow signal reversal in the supraorbital artery. SPP equation accounts for the hemodynamic effect of inflow stenosis and intra-extracranial flow diversion, and is a more precise perfusion pressure target than CPP for the intracranial compartment. Manipulation of intra-extracranial pressure gradient ICP-Pe can augment intracranial inflow pressure (Pd) and reverse intra-extracranial steal.

A stochastic four-state model of contingent gating of gap junction channels containing two "fast" gates sensitive to transjunctional voltage.

Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two "fast" gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (V(j)). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance gamma(h,o) and in the residual state with conductance gamma(h,res), and that both gamma(h,o) and gamma(h,res) rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of V(j) that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by gamma(h,o) and gamma(h,res) rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and V(j)-dependent gating of GJs formed from different connexin isoforms. V(j)-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive V(j) steps rising in amplitude, 2), slowly rising V(j) ramps, and 3), series of V(j) steps of high frequency. The model was used to predict V(j)-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of V(j)-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels.

Application of stochastic automata networks for creation of continuous time Markov chain models of voltage gating of gap junction channels.

The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ~20 times.

Cerebral venous steal: blood flow diversion with increased tissue pressure.

PURPOSE: Flow in areas with increased tissue pressure is described by a Starling resistor and is determined by the inflow pressure (P(i)), the external pressure (P(e)), and the outflow or venous pressure (P(v)). Flow is in Zone 1 at P(e) > P(i) > P(v), Zone 2 at P(i) > P(e) > P(v), and Zone 3 at P(i) > P(v) > P(e). A focal tissue pressure increase after stroke or trauma may lead to a transition from Zone 1 or 2 in the center to Zone 3 in the periphery. We hypothesize that the coexistence of different zones may lead to steal-like blood flow diversion in the perifocal area. CONCEPT: We used a lumped-parameter model of two parallel Starling resistors with a common inflow. The first resistor, with higher P(e), represented the area with increased tissue pressure. The second resistor, with P(e)' = 0, represented the surrounding area. We evaluated the effects of venous pressure on the flow distribution between the two Starling resistors. RATIONALE: The model demonstrated blood flow diversion toward the second Starling resistor with low external pressure. High inflow resistance facilitates this "steal." Flow diversion is caused by effective outflow pressure differences for the Starling resistors (P(e) for the first and P(v) for the second). The venous pressure increase equilibrates the effective backpressure and decreases flow diversion. When the venous pressure equals the external tissue pressure, blood flow diversion (cerebral venous steal) is abolished. Although increased venous pressure causes global flow reduction, it may restore flow to more than 50% of baseline values in areas of increased tissue pressure. DISCUSSION: Cerebral venous steal is a potential cause of secondary brain injury in areas of increased tissue pressure. It can be eliminated with increased venous pressure. Increased venous pressure may recruit the collapsed vascular network and correct perifocal perfusion maldistribution. This resembles how positive end expiratory pressure recruits collapsed airways and decreases the ventilation/perfusion mismatch.

Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45.

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V(j). Cx40/Cx45 heterotypic GJ channels preferentially exhibit V(j)-dependent gating transitions between open and residual states with a conductance of approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.