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 Ca 2+ 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 Ca 2+ 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.

All animals need to sense temperature to avoid hostile environments and to regulate their internal homeostasis. A particularly obvious example is that animals need to avoid damagingly hot stimuli. The mechanisms by which temperature is sensed have until recently been mysterious, but in the last couple of years, we have begun to understand how noxious thermal stimuli are detected by sensory neurons. Heat has been found to open a nonselective cation channel in primary sensory neurons, probably by a direct action. In a separate study, an ion channel gated by capsaicin, the active ingredient of chili peppers, was cloned from sensory neurons. This channel (vanilloid receptor subtype 1, VR1) is gated by heat in a manner similar to the native heat-activated channel, and our current best guess is that this channel is the molecular substrate for the detection of painful heat. Both the heat channel and VR1 are modulated in interesting ways. The response of the heat channel is potentiated by phosphorylation by protein kinase C, whereas VR1 is potentiated by externally applied protons. Protein kinase C is known to be activated by a variety of inf lammatory mediators, including bradykinin, whereas extracellular acidification is characteristically produced by anoxia and inf lammation. Both modulatory pathways are likely, therefore, to have important physiological correlates in terms of the enhanced pain (hyperalgesia) produced by tissue damage and inf lammation. Future work should focus on establishing, in molecular terms, how a single ion channel can detect heat and how the detection threshold can be modulated by hyperalgesic stimuli.Organisms sense temperature for all sorts of reasons. Highly accurate thermosensation is required to set the body temperature of a mammal. Simpler animals sense the external temperature to seek out favorable environments for feeding or for mating. Damaging extremes of temperature must be avoided, of course, and for this purpose, pain-sensitive nerve terminals detect very low and very high temperatures and induce an avoidance response. In all these instances, temperature must be detected-but how? In many cases, the detection mechanism involves a specialized temperature-sensitive nerve terminal, which, on application of a temperature change, generates a depolarization and a resulting train of action potentials in the sensory nerve axon. So it is perhaps obvious to state that temperature must gate an ion channel in the sensory nerve terminal. But how does it work? One could imagine a temperature-sensitive biochemical pathway that modulates an internal transmitter and in turn gates the ion channel. There is some evidence for such a system in at least one temperaturesensitive pathway in the nematode Caenorhabditis elegans (see below). But in the only other instance of which we have any understanding, the action of temperature on the ion channel seems instead to be direct. To the existing voltage-gated, ligand-gated, and mechanosensitive ion channels, we can therefore add a fourth major catego...

The role played by the mechanical tissue stress in supporting lymph formation and propulsion in thoracic tissues was studied in deeply anesthetized rats (n = 13) during spontaneous breathing or mechanical ventilation. After arterial and venous catheterization and insertion of an intratracheal cannula, fluorescent dextrans were injected intrapleurally to serve as lymphatic markers. After 2 h, the fluorescent intercostal lymphatics were identified, and the hydraulic pressure in lymphatic vessels (P lymph) and adjacent interstitial space (P int) was measured using micropuncture. During spontaneous breathing, end-expiratory P lymph and corresponding P int were -2.5 +/- 1.1 (SE) and 3.1 +/- 0.7 mmHg (P < 0.01), which dropped to -21.1 +/- 1.3 and -12.2 +/- 1.3 mmHg, respectively, at end inspiration. During mechanical ventilation with air at zero end-expiratory alveolar pressure, P lymph and P int were essentially unchanged at end expiration, but, at variance with spontaneous breathing, they increased at end inspiration to 28.1 +/- 7.9 and 28.2 +/- 6.3 mmHg, respectively. The hydraulic transmural pressure gradient (DeltaP tm = P lymph - P int) was in favor of lymph formation throughout the whole respiratory cycle (DeltaP tm = -6.8 +/- 1.2 mmHg) during spontaneous breathing but not during mechanical ventilation (DeltaP tm = -1.1 +/- 1.8 mmHg). Therefore, data suggest that local tissue stress associated with the active contraction of respiratory muscles is required to support an efficient lymphatic drainage from the thoracic tissues.

Fluid and solute flux between the pleural and peritoneal cavities, although never documented under physiological conditions, might play a relevant role in pathological conditions associated with the development of ascitis and pleural effusion and/or in the processes of tumor dissemination. To verify whether a pleuroperitoneal flux might take place through the diaphragmatic lymphatic network, the transdiaphragmatic pressure gradient (⌬P TD) was measured in five spontaneously breathing anesthetized rats. ⌬PTD was Ϫ1.93 cmH2O (SD 0.59) and Ϫ3.1 cmH2O (SD 0.82) at end expiration and at end inspiration, respectively, indicating the existence of a pressure gradient directed from the abdominal to the pleural cavity. Morphometrical analysis of the diaphragmatic lymphatic network was performed in the excised diaphragm of three additional rats euthanized with an anesthesia overdose. Optical and electron microscopy revealed that lymphatic submesothelial lacunae and lymphatic capillaries among the skeletal muscles fibers show the ultrastructural features of the socalled initial lymphatic vessels, namely, a discontinuous basal lamina and anchoring filaments linking the outer surface of the endothelial cells to connective tissue or to muscle fibers. Primary unidirectional valves in the wall of the initial lymphatics allow entrance of serosal fluid into the lymphatic network preventing fluid backflow, while unidirectional intraluminar valves in the transverse vessels convey lymph centripetally toward central collecting ducts. The complexity and anatomical arrangement of the two valves system suggests that, despite the existence of a favorable ⌬P TD, in the physiological condition no fluid bulk flow takes place between the pleural and peritoneal cavity through the diaphragmatic lymphatic network. intraluminar lymphatic pressure; serosal fluid pressure; tissue fluid homeostasis THE DRAINAGE OF FLUID, solutes of large molecular weight, and even cells from the pleural and the peritoneal cavity mainly occurs through the lymphatic system located in the parietal mesothelial and submesothelial tissues covering the thoracic and abdominal walls and both surfaces of the diaphragm (10,12,15,19). The ability of the diaphragmatic lymphatic system to drain fluid from both the pleural and peritoneal cavity has been assessed in normal healthy animals (10, 12) by using experimental approaches that were meant to respect the physiological condition as much as possible. The results from these studies, whereas demonstrating the importance of the diaphragmatic lymphatics in maintaining the serosal fluid volume, failed to reveal or suggest the occurrence of fluid transfer between the pleural and peritoneal cavities through the diaphragm itself. However, the existence of direct transdiaphragmatic lymphatic pathways often has been proposed (4, 25) to explain clinical observations like the development of hydrothoraces secondary to peritoneal dialysis or ascitis. At present it is not clear whether the recruitment of a direct transdiaphragmatic pathway wi...

The data revealed a great functional complexity of the diaphragmatic lymphatic network and suggested that cardiogenic oscillations may play an important role in promoting lymph formation and propulsion from interstitial tissues with subatmospheric tissue pressure.

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.

This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V(T)) might affect the composition and/or structure of the glycosaminoglycan (GAG) components of pulmonary extracellular proteoglycans. Experiments were performed in 30 healthy rats: 1) anesthetized and immediately killed (controls, C-0); 2) anesthetized and spontaneously breathing for 4 h (C-4h); and 3) anesthetized, paralyzed, and mechanically ventilated for 4 h with air at 0-cmH(2)O end-expiratory pressure and V(T) of 8 ml/kg (MV-1), 16 ml/kg (MV-2), 24 ml/kg (MV-3), or 32 ml/kg (MV-4), adjusting respiratory rates at a minute ventilation of 270 ml/min. Compared with C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, MV induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bound GAGs and wash out of loosely bound or fragmented GAGs progressively increased with increasing V(T) and was associated with increased expression of local (matrix metalloproteinase-2) and systemic (matrix metalloproteinase-9) activated metalloproteases. We conclude that 1) MV, even at "physiological" low V(T), severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; and 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.

Neuronal and inducible nitric oxide synthase (nNOS and iNOS) play a protective and damaging role, respectively, on the intestinal neuromuscular function after ischemia-reperfusion (I/R) injury. To uncover the molecular pathways underlying this dichotomy we investigated their possible correlation with the orthodenticle homeobox proteins OTX1 and OTX2 in the rat small intestine myenteric plexus after in vivo I/R. Homeobox genes are fundamental for the regulation of the gut wall homeostasis both during development and in pathological conditions (inflammation, cancer). I/R injury was induced by temporary clamping the superior mesenteric artery under anesthesia, followed by 24 and 48 h of reperfusion. At 48 h after I/R intestinal transit decreased and was further reduced by -propyl-l-arginine hydrochloride (NPLA), a nNOS-selective inhibitor. By contrast this parameter was restored to control values by 1400W, an iNOS-selective inhibitor. In longitudinal muscle myenteric plexus (LMMP) preparations, iNOS, OTX1, and OTX2 mRNA and protein levels increased at 24 and 48 h after I/R. At both time periods, the number of iNOS- and OTX-immunopositive myenteric neurons increased. nNOS mRNA, protein levels, and neurons were unchanged. In LMMPs, OTX1 and OTX2 mRNA and protein upregulation was reduced by 1400W and NPLA, respectively. In myenteric ganglia, OTX1 and OTX2 staining was superimposed with that of iNOS and nNOS, respectively. Thus in myenteric ganglia iNOS- and nNOS-derived NO may promote OTX1 and OTX2 upregulation, respectively. We hypothesize that the neurodamaging and neuroprotective roles of iNOS and nNOS during I/R injury in the gut may involve corresponding activation of molecular pathways downstream of OTX1 and OTX2. Intestinal ischemia-reperfusion (I/R) injury induces relevant alterations in myenteric neurons leading to dismotility. Nitrergic neurons seem to be selectively involved. In the present study the inference that both neuronal and inducible nitric oxide synthase (nNOS and iNOS) expressing myenteric neurons may undergo important changes sustaining derangements of motor function is reinforced. In addition, we provide data to suggest that NO produced by iNOS and nNOS regulates the expression of the vital transcription factors orthodenticle homeobox protein 1 and 2 during an I/R damage.