Andrea Moriondo

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

1 The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2 Maximal activation of PKC by a brief application of phorbol 12‐myristate 13‐acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65‐fold in DRG neurones and 2.18‐fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31‐8220 prevented the enhancement caused by PMA. 3 Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4 Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5 Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40‐fold in DRG neurones and 3.42‐fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6 The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin‐gated current. 7 The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium‐dependent process.

Lymphatic anatomy and biomechanics

Abstract  Lymph formation is driven by hydraulic pressure gradients developing between the interstitial tissue and the lumen of initial lymphatics. While in vessels equipped with lymphatic smooth muscle cells these gradients are determined by well‐synchronized spontaneous contractions of vessel segments, initial lymphatics devoid of smooth muscles rely on tissue motion to form lymph and propel it along the network. Lymphatics supplying highly moving tissues, such as skeletal muscle, diaphragm or thoracic tissues, undergo cyclic compression and expansion of their lumen imposed by local stresses arising in the tissue as a consequence of cardiac and respiratory activities. Active muscle contraction and not passive tissue displacement is required to support an efficient lymphatic drainage, as suggested by the fact that the respiratory activity promotes lymph formation during spontaneous, but not mechanical ventilation. The mechanical properties of the lymphatic wall and of the surrounding tissue also play an important role in lymphatic function. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels behave as reservoirs accommodating absorbed interstitial fluid, while lymphatics with stiffer walls, taking advantage of a more efficient transmission of tissue stresses to the lymphatic lumen, propel fluid through the lumen of the lymphatic circuit.

Calcium‐activated potassium current clamps the dark potential of vertebrate rods

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of ≈ −35 mV. The present work investigates the physiological role of the Ca2+‐activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light‐ and dark‐adapted rods under voltage‐ or current‐clamp conditions, respectively. In light‐adapted rods of the salamander, selective blockade of Ca2+‐activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current‐clamped rods by ≈ 30 mV, from a resting potential of ≈ −35 mV. A similar depolarization was observed if external Ca2+ (1 mm) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to −100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of ≈ −20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark‐adapted current‐clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady‐state depolarizations of ≈ 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+‐activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.

Pleural function and lymphatics.

The pleural space plays an important role in respiratory function as the negative intrapleural pressure regimen ensures lung expansion and in the mean time maintains the tight mechanical coupling between the lung and the chest wall. The efficiency of the lung–chest wall coupling depends upon pleural liquid volume, which in turn reflects the balance between the filtration of fluid into and its egress out of the cavity. While filtration occurs through a single mechanism passively driving fluid from the interstitium of the parietal pleura into the cavity, several mechanisms may co‐operate to remove pleural fluid. Among these, the pleural lymphatic system emerges as the most important one in quantitative terms and the only one able to cope with variable pleural fluid volume and drainage requirements. In this review, we present a detailed account of the actual knowledge on: (a) the complex morphology of the pleural lymphatic system, (b) the mechanism supporting pleural lymph formation and propulsion, (c) the dependence of pleural lymphatic function upon local tissue mechanics and (d) the effect of lymphatic inefficiency in the development of clinically severe pleural and, more in general, respiratory pathologies.

Impact of mechanical ventilation and fluid load on pulmonary glycosaminoglycans.

The combined effect of mechanical ventilation and fluid load on pulmonary glycasaminoglycans (GAGs) was studied in anaesthetized rats ((BW 290±21.8 (SE)g) mechanically ventilated for 4h: (a) at low (∼7.5mlkg(-1)) or high (∼23mlkg(-1)) tidal volume (V(T)) and zero alveolar pressure; (b) at low or high V(T) at 5cmH(2)O positive end-expiratory pressure (PEEP); (c) with or without 7mlkg(-1)h(-1) intravenous infusion of Phosphate Buffer Solution (PBS). Compared to spontaneous breathing, GAGs extractability decreased by 52.1±1.5% and 42.2±7.3% in not-infused lungs mechanically ventilated at low V(T) or at high V(T) and PEEP, respectively. In contrast, in infused lungs, GAGs extractability increased by 56.1±4.0% in spontaneous ventilation and PEEP and up to 81.1% in all mechanically ventilated lungs, except at low V(T) without PEEP. In the absence of an inflammatory process, these results suggest that PEEP was protective at low but not at high V(T) when alveolar structures experience exceedingly high stresses. When combined to mechanical ventilation, fluid load might exacerbate edema development and lung injury.

Tissue contribution to the mechanical features of diaphragmatic initial lymphatics

The role of the mechanical properties of the initial lymphatic wall and of the surrounding tissue in supporting lymph formation and/or progression was studied in six anaesthetized, neuromuscularly blocked and mechanically ventilated rats. After mid‐sternal thoracotomy, submesothelial initial lymphatics were identified on the pleural diaphragmatic surface through stereomicroscopy. An ‘in vivo’ lymphatic segment was prepared by securing two surgical threads around the vessel at a distance of ∼2.5 mm leaving the vessel in place. Two glass micropipettes were inserted into the lumen, one for intraluminar injections of 4.6 nl saline boluses and one for hydraulic pressure (Plymph) recording. The compliance of the vessel wall (Clymph) was calculated as the slope of the plot describing the change in segment volume as a function of the post‐injection Plymph changes. Two superficial lymphatic vessel populations with a significantly different Clymph (6.7 ± 1.6 and 1.5 ± 0.4 nl mmHg−1 (mean ±s.e.m.), P < 0.001) were identified. In seven additional rats, the average elastic modulus of diaphragmatic tissue strips was determined by uniaxial tension tests to be 1.7 ± 0.3 MPa. Clymph calculated for an initial lymphatic completely surrounded by isotropic tissue was 0.068 nl mmHg−1, i.e. two orders of magnitude lower than in submesothelial lymphatics. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels may act as reservoirs accommodating large absorbed fluid volumes, while lymphatics with stiffer walls serve to propel fluid through the lumen of the lymphatic vessel by taking advantage of the more efficient mechanical transmission of tissue stresses to the lymphatic lumen.

Vertebrate rod photoreceptors express both BK and IK calcium-activated potassium channels, but only BK channels are involved in receptor potential regulation.

In salamander rods, Ca2+‐activated K+ current (IKCa) provides an effective “clamp” of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large‐conductance Ca2+‐ and voltage‐dependent potassium (BK) channel and intermediate‐conductance Ca2+‐dependent potassium (IK) channel, but not small‐conductance Ca2+‐dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net IKCa to 36% and 63%, respectively, whereas the current was unaffected by application of 1 μM apamin. Consistently, anti‐ SK1, ‐SK2, and ‐SK3 antibodies were unable to stain rod photoreceptors, whereas both anti‐BK and ‐SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current‐clamp experiments, it was clearly seen that the strong clamping effect of the total IKCa was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

Kinetics of fluid flux in the rat diaphragmatic submesothelial lymphatic lacunae

The specific role of loops and/or linear segments in pleural diaphragmatic submesothelial lymphatics was investigated in seven anesthetized, paralyzed, and mechanically ventilated rats. Lymphatic loops lay peripherally above the diaphragmatic muscular plane, whereas linear vessels run over both the muscular and central tendineous regions. Lymph vessel diameter, measured by automatic software analysis, was significantly greater (P < 0.01) in linear vessels [103.4 +/- 8.5 microm (mean +/- SE), n = 18] than in loops (54.6 +/- 3.3 microm, n = 21). Conversely, the geometric mean of intraluminal flow velocity, obtained from the speed of distribution of a bolus of fluorescent dextrans injected into the vessel, was lower (P < 0.01) in linear vessels (26.3 +/- 1.4 microm/s) compared with loops (51.3 +/- 3.2 microm/s). Lymph flow, calculated as the product of flow velocity by vessel cross-sectional area, was similar in linear vessels and in individual vessels of a loop, averaging 8.6 +/- 1.6 nl/min. Flow was always directed from the diaphragm periphery toward the medial tendineous region in linear vessels, whereas it was more complex and evidently controlled by intraluminal unidirectional valves in loops. The results suggest that loops might be the preferential site of lymph formation, whereas linear vessels would be mainly involved in the progression of newly formed lymph toward deeper collecting diaphragmatic ducts. Within the same hierarchic order of diaphragmatic lymphatic vessels, the spatial organization and geometrical arrangement of the submesothelial lacunae seem to be finalized at exploiting the alternate contraction/relaxation phases of diaphragmatic muscle fibers to optimize fluid removal from serosal cavities.

A step-by-step model of phototransduction cascade shows that Ca2+ regulation of guanylate cyclase accounts only for short-term changes of photoresponse

A mathematical model of the vertebrate phototransduction mechanism was designed in a modular fashion, in that increasingly complex behaviors can be turned on and off to evaluate the relative involvement of all elements of the phototransduction cascade. The problem was approached by starting with a minimum model in which the intracellular cGMP concentration ([cGMP]i) was determined by guanylate cyclase (GC), whose activity was assumed not to be regulated by any factor (such as Ca2+) and by phosphodiesterase (PDE), whose activity was assumed to be proportional to the light intensity. All dependences were subsequently introduced, i.e. the equations describing PDE activation in detail, the Ca2+ regulation of GC and the action of intracellular Ca2+ buffers. The simulations and fits show that a high-gain, smooth time- and light-dependent PDE activation, a Ca2+-dependent GC, and a Ca2+-dependent buffer mechanism are required to account for the flash response kinetics in the dark and on dim backgrounds of light, and the effect of exogenous Ca2+ buffers to produce responses characterized by slow and damped oscillations and to enhance the low-frequency noise. However, it was not possible to find any set of parameters able to simultaneously interpolate the waveform of the flash responses (in the dark and on a background of light) and the responses to steps of light. It is therefore concluded that at least one more shut-off mechanism (possibly not Ca-dependent) is necessary to fully account for the phenomenology of the light response in rod photoreceptors.

Impact of respiratory pattern on lung mechanics and interstitial proteoglycans in spontaneously breathing anaesthetized healthy rats.

Aim:  The aim of this study was to investigate the effect of different pattern of spontaneous breathing on the respiratory mechanics and on the integrity of the pulmonary extracellular matrix.