Impact of chemical modification of sulfamidase on distribution to brain interstitial fluid and to CSF after an intravenous administration in awake, freely-moving rats.

Mucopolysaccharidosis III A (MPS IIIA) is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. The disorder results in accumulation of heparan sulfate, lysosomal enlargement and cellular and organ dysfunction. Patients exhibit progressive neurodegeneration and behavioral problems and no treatment is currently available. Enzyme replacement therapy is explored as potential treatment strategy for MPS IIIA patients and to modify the disease, sulfamidase must reach the brain. The glycans of recombinant human sulfamidase (rhSulfamidase) can be chemically modified to generate CM-rhSulfamidase. The chemical modification reduced the affinity to the cation-independent mannose-6-phosphate receptor with the aim a prolonged higher concentration in circulation and thus at the blood brain barrier. The pharmacokinetic properties in serum and the distribution to brain and to cerebrospinal fluid (CSF) of chemically modified recombinant human sulfamidase (CM-rhSulfamidase) were studied and compared to those of rhSulfamidase, after a single intravenous (i.v.) 30 mg/kg dose in awake, freely-moving male Sprague Dawley rats. Distribution to brain was studied by microdialysis of the interstitial fluid in prefrontal cortex and by repeated intra-individual CSF sampling from the cisterna magna. Push-pull microdialysis facilitated sampling of brain interstitial fluid to determine large molecule concentrations in awake, freely-moving male Sprague Dawley rats. Together with repeated serum and CSF sampling, push-pull microdialysis facilitated determination of CM-rhSulfamidase and rhSulfamidase kinetics after i.v. administration by non-compartments analysis and by a population modelling approach. Chemical modification increased the area under the concentration versus time in serum, CSF and brain interstitial fluid at least 7-fold. The results and the outcome of a population modelling approach of the concentration versus time data indicated that both compounds pass the BBB with an equilibrium established fairly rapid after administration. We suggest that prolonged high serum concentrations facilitated high brain interstitial fluid concentrations, which could be favorable to reach various target cells in the brain.

Loss of intestinal sympathetic innervation elicits an innate immune driven colitis.

BACKGROUND: Both the parasympathetic and sympathetic nervous system exert control over innate immune responses. In inflammatory bowel disease, sympathetic innervation in intestinal mucosa is reduced. Our aim was to investigate the role of sympathetic innervation to the intestine on regulation of the innate immune responses. METHODS: In lipopolysaccharide (LPS)-stimulated macrophages, we evaluated the effect of adrenergic receptor activation on cytokine production and metabolic profile. In vivo, the effect of sympathetic denervation on mucosal innate immune responses using 6-hydroxydopamine (6-OHDA), or using surgical transection of the superior mesenteric nerve (sympathectomy) was tested in Rag1-/- mice that lack T- and B-lymphocytes. RESULTS: In murine macrophages, adrenergic ß2 receptor activation elicited a dose-dependent reduction of LPS-induced cytokines, reduced LPS-induced glycolysis and increased maximum respiration. Sympathectomy led to a significantly decreased norepinephrine concentration in intestinal tissue. Within 14 days after sympathectomy, mice developed clinical signs of colitis, colon oedema and excess colonic cytokine production. Both 6-OHDA and sympathectomy led to prominent goblet cell depletion and histological damage of colonic mucosa. CONCLUSIONS: We conclude that the sympathetic nervous system plays a regulatory role in constraining innate immune cell reactivity towards microbial challenges, likely via the adrenergic ß2 receptor.

Development of a Rat Plasma and Brain Extracellular Fluid Pharmacokinetic Model for Bupropion and Hydroxybupropion Based on Microdialysis Sampling, and Application to Predict Human Brain Concentrations.

Administration of bupropion [(±)-2-(tert-butylamino)-1-(3-chlorophenyl)propan-1-one] and its preformed active metabolite, hydroxybupropion [(±)-1-(3-chlorophenyl)-2-[(1-hydroxy-2-methyl-2-propanyl)amino]-1-propanone], to rats with measurement of unbound concentrations by quantitative microdialysis sampling of plasma and brain extracellular fluid was used to develop a compartmental pharmacokinetics model to describe the blood-brain barrier transport of both substances. The population model revealed rapid equilibration of both entities across the blood-brain barrier, with resultant steady-state brain extracellular fluid/plasma unbound concentration ratio estimates of 1.9 and 1.7 for bupropion and hydroxybupropion, respectively, which is thus indicative of a net uptake asymmetry. An overshoot of the brain extracellular fluid/plasma unbound concentration ratio at early time points was observed with bupropion; this was modeled as a time-dependent uptake clearance of the drug across the blood-brain barrier. Translation of the model was used to predict bupropion and hydroxybupropion exposure in human brain extracellular fluid after twice-daily administration of 150 mg bupropion. Predicted concentrations indicate that preferential inhibition of the dopamine and norepinephrine transporters by the metabolite, with little to no contribution by bupropion, would be expected at this therapeutic dose. Therefore, these results extend nuclear imaging studies on dopamine transporter occupancy and suggest that inhibition of both transporters contributes significantly to bupropion's therapeutic efficacy.

Glutamatergic regulation of brain histamine neurons: In vivo microdialysis and electrophysiology studies in the rat.

The interactions between the glutamatergic and the histaminergic systems in the brain are not fully understood. Here we studied histamine release in the medial prefrontal cortex and the posterior hypothalamus-tuberomamillary nucleus (PH-TMN) using in vivo microdialysis and electrophysiological recordings of histaminergc neurons in the PH-TMN in vivo to further address the mechanistic details of these interactions. We demonstrated that histaminergic activity was regulated by group II metabotropic glutamate receptors (mGluR 2 and 3) using systemic dosing with mGluR 2/3 agonist and antagonists and an mGluR 2 positive allosteric modulator. These interactions likely occur via direct modulation of glutamate release in the PH-TMN. The importance of circadian rhythm for histamine release was also shown using microdialysis studies with mGluR 2/3 compounds under light and dark conditions. Based on histamine release studies with NMDA and ketamine, we propose the existence of two sub-populations of NMDA receptors where one subtype is located on histaminergic cell bodies in the PH-TMN and the second on GABA-ergic neurons projecting to the PH-TMN. These subpopulations could be distinguished based on function, notably opposing actions were seen on histamine release in the medial prefrontal cortex of the rat. In summary, this paper provides evidence that the histaminergic system is closely regulated by glutamate neurons in multiple ways. In addition, this interaction depends to a great extent on the activity state of the subject.

Effects of lisdexamfetamine alone and in combination with s-citalopram on acetylcholine and histamine efflux in the rat pre-frontal cortex and ventral hippocampus.

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by poor attention, impulse control and hyperactivity. A significant proportion of ADHD patients are also co-morbid for other psychiatric problems including mood disorders and these patients may be managed with a combination of psychostimulants and anti-depressants. While it is generally accepted that enhanced catecholamine signalling via the action of psychostimulants is likely responsible for the cognitive improvement in ADHD, other neurotransmitters including acetylcholine and histamine may be involved. In the present study, we have examined the effect of lisdexamfetamine dimesylate (LDX), an amphetamine pro-drug that is approved for the treatment of ADHD on acetylcholine and histamine efflux in pre-frontal cortex and hippocampus alone and in combination with the anti-depressant s-citalopram. LDX increased cortical acetylcholine efflux, an effect that was not significantly altered by co-administration of s-citalopram. Cortical and hippocampal histamine were markedly increased by LDX, an effect that was attenuated in the hippocampus but not in pre-frontal cortex when co-administered with s-citalopram. Taken together, these results suggest that efflux of acetylcholine and histamine may be involved in the therapeutic effects of LDX and are differentially influenced by the co-administration of s-citalopram. Attention deficit hyperactivity disorder (ADHD) is characterized by poor attention, impulse control and hyperactivity. Some ADHD patients are also co-morbid for mood disorders and may be managed with psychostimulants (e.g. lisdexamfetamine, LDX) and anti-depressants (e.g. s-citalopram). LDX increased the efflux of acetylcholine and histamine, neurotransmitters involved in cognitive function, which were differentially influenced when co-administered with s-citalopram. Acetylcholine and histamine may be involved in the therapeutic effects of LDX and are differentially affected by the co-administration of s-citalopram.

Interaction Between Brain Histamine and Serotonin, Norepinephrine, and Dopamine Systems: In Vivo Microdialysis and Electrophysiology Study.

Brain monoamines (serotonin, norepinephrine, dopamine, and histamine) play an important role in emotions, cognition, and pathophysiology and treatment of mental disorders. The interactions between serotonin, norepinephrine, and dopamine were studied in numerous works; however, histamine system received less attention. The aim of this study was to investigate the interactions between histamine and other monoamines, using in vivo microdialysis and electrophysiology. It was found that the inverse agonist of histamine-3 receptors, thioperamide, increased the firing activity of dopamine neurons in the ventral tegmental area. Selective agonist of histamine-3 receptors, immepip, reversed thiperamide-induced stimulation of firing activity of dopamine neurons. The firing rates of serotonin and norpeinephrine neurons were not attenuated by immepip or thioperamide. Thioperamide robustly and significantly increased extracellular concentrations of serotonin, norepinephrine, and dopamine in the rat prefrontal cortex and slightly increased norepinephrine and dopamine levels in the tuberomammillary nucleus of the hypothalamus. It can be concluded that histamine stimulates serotonin, norepinephrine, and dopamine transmission in the brain. Modulation of firing of dopamine neurons is a key element in functional interactions between histamine and other monoamines. Antagonists of histamine-3 receptors, because of their potential ability to stimulate monoamine neurotransmission, might be beneficial in the treatment of mental disorders.

Upregulation of the dorsal raphe nucleus-prefrontal cortex serotonin system by chronic treatment with escitalopram in hyposerotonergic Wistar-Kyoto rats.

Wistar-Kyoto (WKY) rats are sensitive to chronic stressors and exhibit depression-like behavior. Dorsal raphe nucleus (DRN) serotonin (5-HT) neurons projecting to the prefrontal cortex (PFC) comprise the important neurocircuitry underlying the pathophysiology of depression. To evaluate the DRN-PFC 5-HT system in WKY rats, we examined the effects of escitalopram (ESCIT) on the extracellular 5-HT level in comparison with Wistar rats using dual-probe microdialysis. The basal levels of 5-HT in the DRN, but not in the PFC, in WKY rats was reduced as low as 30% of Wistar rats. Responses of 5-HT in the DRN and PFC to ESCIT administered systemically and locally were attenuated in WKY rats. Feedback inhibition of DRN 5-HT release induced by ESCIT into the PFC was also attenuated in WKY rats. Chronic ESCIT induced upregulation of the DRN-PFC 5-HT system in WKY rats, with increases in basal 5-HT in the DRN, responsiveness to ESCIT in the DRN and PFC, and feedback inhibition, whereas downregulation of these effects was induced in Wistar rats. Thus, the WKY rat is an animal model of depression with low activity of the DRN-PFC 5HT system. The finding that chronic ESCIT upregulates the 5-HT system in hyposerotonergic WKY rats may contribute to improved understanding of mechanisms of action of antidepressants, especially in depression with 5-HT deficiency.

Mechanoprotection by polycystins against apoptosis is mediated through the opening of stretch-activated K(2P) channels.

How renal epithelial cells respond to increased pressure and the link with kidney disease states remain poorly understood. Pkd1 knockout or expression of a PC2 pathogenic mutant, mimicking the autosomal dominant polycystic kidney disease, dramatically enhances mechanical stress-induced tubular apoptotic cell death. We show the presence of a stretch-activated K(+) channel dependent on the TREK-2 K(2P) subunit in proximal convoluted tubule epithelial cells. Our findings further demonstrate that polycystins protect renal epithelial cells against apoptosis in response to mechanical stress, and this function is mediated through the opening of stretch-activated K(2P) channels. Thus, to our knowledge, we establish for the first time, both in vitro and in vivo, a functional relationship between mechanotransduction and mechanoprotection. We propose that this mechanism is at play in other important pathologies associated with apoptosis and in which pressure or flow stimulation is altered, including heart failure or atherosclerosis.

Tapentadol increases levels of noradrenaline in the rat spinal cord as measured by in vivo microdialysis.

Spinal noradrenaline is thought to play an important role in descending pain inhibitory pathways and the modulation of nociceptive information at the spinal level. Tapentadol is a µ-opioid receptor (MOR) agonist and noradrenaline reuptake inhibitor (NRI). We showed previously that tapentadol, in contrast to morphine, elevates levels of noradrenaline, but not serotonin, in the ventral hippocampus of rats. The aim of this study was to examine the effects of tapentadol, morphine and venlafaxine on spinal monoamine levels. Rats were implanted with spinal microdialysis probes. Drugs were administered intraperitoneally, and samples were collected for 3h in isoflurane-anesthetized animals and analysed for monoamine content using HPLC-MS/MS. In terms of area-under-curve (AUC, 0-180 min), tapentadol (4.64-21.5mg/kg) produced a dose-dependent, significant increase in extracellular spinal noradrenaline levels (9275±4346 min% at the highest dose versus -1047±889 min% for vehicle). A maximum increase of 182±32% of baseline was reached 60 min after administration of 10mg/kg tapentadol. Venlafaxine (10mg/kg) produced an effect of similar magnitude. In contrast, tapentadol decreased extracellular spinal serotonin levels (non-significantly compared to vehicle), while venlafaxine increased spinal serotonin to 267±74% of baseline. In contrast to tapentadol and venlafaxine, morphine slightly decreased levels of noradrenaline and serotonin. This study demonstrates that analgesic doses of tapentadol (and venlafaxine), but not morphine, increase spinal noradrenaline levels and that tapentadol is devoid of a relevant serotonergic effect. It supports the suggestion that the NRI component of tapentadol is functionally relevant and contributes to its mechanism of action.

The role of cortical and hypothalamic histamine-3 receptors in the modulation of central histamine neurotransmission: an in vivo electrophysiology and microdialysis study.

The current study aimed to investigate the effect of histamine-3 (H(3)) receptors, expressed in the tuberomammillary nucleus (TMN) of the hypothalamus and in the prefrontal cortex (PFC), on histamine neurotransmission in the rat brain. The firing activity of histamine neurons in the TMN was measured using in vivo extracellular single-unit electrophysiology, under propofol anesthesia. Extracellular histamine levels were determined using the dual (PFC and TMN) probe microdialysis, in freely-moving animals. Histamine levels in dialysates were determined using high-performance liquid chromatography (HPLC) and fluorescence detection. It was found that systemic administration of the selective H(3)-agonist, immepip, decreases, and the reverse H(3) /H(4)-agonist, thioperamide, increases the firing activity of histamine neurons in the TMN and the release of histamine in TMN and PFC. Local perfusion of immepip into the TMN increased, and thioperamide decreased, histamine levels in the TMN but not in the PFC. Local perfusion of immepip into the PFC, however, decreased extracellular histamine levels in both TMN and PFC. It can be concluded that brain H(3) receptors, and especially those expressed in the PFC, play an important role in the autoregulation of histamine neurotransmission. It is possible that H(3) receptors in the PFC are expressed on pyramidal neurons projecting to the TMN, and activation of these receptors diminishes glutamate excitatory input from PFC to the TMN. As the brain histamine system has a role in pathophysiology of psychotic, affective, cognitive, sleep and eating disorders, H(3) receptors are potential targets for future CNS medications.

Effect of co-administration of varenicline and antidepressants on extracellular monoamine concentrations in rat prefrontal cortex.

Since a substantial proportion of smokers have comorbid mood disorders, the smoking cessation aid varenicline might occasionally be prescribed to patients who are simultaneously treated with antidepressants. Given that varenicline is a selective nicotinic acetylcholine receptor partial agonist and not a substrate or inhibitor of drug metabolizing enzymes, pharmacokinetic interactions with various classes of antidepressants are highly unlikely. It is, however, conceivable that varenicline may have a pharmacodynamic effect on antidepressant-evoked increases in central monoamine release. Interactions resulting in excessive transmitter release could cause adverse events such as serotonin syndrome, while attenuation of monoamine release could impact the clinical efficacy of antidepressants. To investigate this we examined whether varenicline administration modulates the effects of the selective serotonin reuptake inhibitor sertraline and the monoamine oxidase inhibitor clorgyline, given alone and combined, on extracellular concentrations of the monoamines serotonin, dopamine, and norepinephrine in rat brain by microdialysis. Given the important role attributed to cortical monoamine release in serotonin syndrome as well as antidepressant activity, the effects on extracellular monoamine concentrations were measured in the medial prefrontal cortex. Responses to maximally effective doses of sertraline or clorgyline and of sertraline plus clorgyline were the same in the absence as in the presence of a relatively high dose of varenicline, which by itself had no significant effect on cortical monoamine release. This is consistent with the binding profile of varenicline that has insufficient affinity for receptors, enzymes, or transporters to inhibit or potentiate the pharmacologic effects of antidepressants. Since varenicline neither diminished nor potentiated sertraline- or clorgyline-induced increases in neurotransmitter levels, combining varenicline with serotonergic antidepressants is unlikely to cause excessive serotonin release or to attenuate antidepressant efficacy via effects on cortical serotonin, dopamine or norepinephrine release.

Sensing pressure in the cardiovascular system: Gq-coupled mechanoreceptors and TRP channels.

Despite the central physiological importance of cardiovascular mechanotransduction, the molecular identities of the sensors and the signaling pathways have long remained elusive. Indeed, how pressure is transduced into cellular excitation has only recently started to emerge. In both arterial and cardiac myocytes, the diacylglycerol-sensitive canonical transient receptor potential (TRPC) subunits are proposed to underlie the stretch-activated depolarizing cation channels. An indirect mechanism of activation through a ligand-independent conformational switch of Gq-coupled receptors by mechanical stress is invoked. Such a mechanism involving the angiotensin type 1 receptor and TRPC6 is proposed to trigger the arterial myogenic response to intraluminal pressure. TRPC6 is also involved in load-induced cardiac hypertrophy. In this review, we will focus on the molecular basis of pressure sensing in the cardiovascular system and associated disease states.

Polycystin-1 and -2 dosage regulates pressure sensing.

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.

Relevance of dorsal raphe nucleus firing in serotonin 5-HT(2C) receptor blockade-induced augmentation of SSRIs effects.

Selective serotonin reuptake inhibitors are the most widely prescribed antidepressant drugs. However, they exhibit a slow onset of action, putatively due to the initial decrease in serotonin cell firing mediated via somato-dendritic autoreceptors. Interestingly, blockade of 5-HT(2C) receptors significantly potentiates the effect of citalopram, a selective serotonin reuptake inhibitor, on serotonin efflux in the hippocampus and prefrontal cortex (Cremers, T.I.F.H., Giorgetti, M., Bosker, F.J., Hogg, S., Arnt, J., Mork, A., Honig, G., Bøgesø, K.P., Westerink, B.H.C., den Boer, J.A., Wikstrøm, H.V., Tecott, L.H., 2004. Inactivation of 5-HT(2C) receptors potentiates consequences of serotonin reuptake blockade. Neuropsychopharmacology 29, 1782-1789; Cremers, T.I.F.H., Rea, K., Bosker, F.J., Wikström, H.V., Hogg, S., Mørk, A., Westerink, B.H.C., 2007. Augmentation of SSRI effects on serotonin by 5-HT(2C) antagonists: mechanistic studies. Neuropsychopharmacology 32, 1550-1557.). Using in vivo electrophysiology, we show in the present study that the purported selective 5-HT(2C) receptor antagonist, SB242,084, dose-dependently counteracts citalopram-induced inhibition of serotonin cell firing. Even though the effect of SB242,084 is significant at a dose found in vivo to also partially occupy 5-HT(2A) receptors, indicating a possible contribution of a partial blockade of 5-HT(2A) receptors together with 5-HT(2C) receptors, we suggest that high occupancy at 5-HT(2C) receptors is essential for the blockade of the inhibitory effect of citalopram on 5-HT cell firing. Using microdialysis, we also show that the potentiation by SB242,084 on serotonin efflux requires an action of citalopram outside the terminal, most likely at the somato-dendritic level (i.e., on serotonin cell firing). Further experiments using local 5-HT(2C) receptor blockade indicate a role of 5-HT(2C) receptors located in the prefrontal cortex. Modulation of short or long feedback loops originating in the prefrontal cortex by 5-HT(2C) receptors could directly inhibit serotonin efflux, or alternatively, regulate serotonin cell firing in the dorsal raphe nucleus, thereby modulating serotonin efflux indirectly.

Antagonism of 5-HT(1A) receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT(7) receptors.

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5-HT) receptor subtype next to the 5-HT(1A) autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5-HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5-HT(7) receptors. Experiments were performed with the specific 5-HT(7) antagonist SB 258741 and the putative 5-HT(7) agonist AS19. In this study WAY 100.635 was used to block 5-HT(1A) receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5-HT in the ventral hippocampus as well as 5-HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co-administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5-HT in ventral hippocampus, hinting at opposed 5-HT(7) receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell-shaped dose-effect curve: moderately increasing 5-HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5-HT(7) receptors and moderate affinity for 5-HT(1A) receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5-HT neuronal activity mediated by 5-HT(7) receptors. It can be speculated, that the restoration of 5-HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5-HT(1A) receptors alone, in fact results from a shift in balance between 5-HT(1A) and 5-HT(7) receptor function.

The mechano-gated K(2P) channel TREK-1.

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K+ channels. More than 80 genes encoding the main K+ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K+ channels. Mammalian two-pore domain K+ channels (K(2P)) make up one class of K+ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K+ channels. Each K(2P) channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including "background" or "leak" K+ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K(2P) channel TREK-1 and will emphasize on the polymodal function of this "unconventional" K+ channel.

Molecular basis of the mammalian pressure-sensitive ion channels: focus on vascular mechanotransduction.

Mechano-gated ion channels are implicated in a variety of neurosensory functions ranging from touch sensitivity to hearing. In the heart, rhythm disturbance subsequent to mechanical effects is also associated with the activation of stretch-sensitive ion channels. Arterial autoregulation in response to hemodynamic stimuli, a vital process required for protection against hypertension-induced injury, is similarly dependent on the activity of force-sensitive ion channels. Seminal work in prokaryotes and invertebrates, including the nematode Caenorhabditis elegans and the fruit fly drosophila, greatly helped to identify the molecular basis of volume regulation, hearing and touch sensitivity. In mammals, more recent findings have indicated that members of several structural family of ion channels, namely the transient receptor potential (TRP) channels, the amiloride-sensitive ENaC/ASIC channels and the potassium channels K2P and Kir are involved in cellular mechanotransduction. In the present review, we will focus on the molecular and functional properties of these channel subunits and will emphasize on their role in the pressure-dependent arterial myogenic constriction and the flow-mediated vasodilation.

TRP channels and mechanosensory transduction: insights into the arterial myogenic response.

Mechano-gated ion channels are implicated in a variety of key physiological functions ranging from touch sensitivity to arterial pressure regulation. Seminal work in prokaryotes and invertebrates provided strong evidence for the role of specific ion channels in volume regulation, touch sensitivity, or hearing, specifically the mechanosensitive channel subunits of large and small conductances (MscL and MscS), the mechanosensory channel subunits (MEC) and the transient receptor potential channel subunits (TRP). In mammals, recent studies further indicate that members of the TRP channel family may also be considered as possible candidate mechanosensors responding to either tension, flow, or changes in cell volume. However, contradictory results have challenged whether these TRP channels, including TRPC1 and TRPC6, are directly activated by mechanical stimulation. In the present review, we will focus on the mechanosensory function of TRP channels, discuss whether a direct or indirect mechanism is at play, and focus on the proposed role for these channels in the arterial myogenic response to changes in intraluminal pressure.

Engineering covalent oligomers of the mechanosensitive channel of large conductance from Escherichia coli with native conductance and gating characteristics.

To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild-type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild-type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.

Lactococcus lactis uses MscL as its principal mechanosensitive channel.

The functions of the mechanosensitive channels from Lactococcus lactis were determined by biochemical, physiological, and electrophysiological methods. Patch-clamp studies showed that the genes yncB and mscL encode MscS and MscL-like channels, respectively, when expressed in Escherichia coli or if the gene products were purified and reconstituted in proteoliposomes. However, unless yncB was expressed in trans, wild type membranes of L. lactis displayed only MscL activity. Membranes prepared from an mscL disruption mutant did not show any mechanosensitive channel activity, irrespective of whether the cells had been grown on low or high osmolarity medium. In osmotic downshift assays, wild type cells survived and retained 20% of the glycine betaine internalized under external high salt conditions. On the other hand, the mscL disruption mutant retained 40% of internalized glycine betaine and was significantly compromised in its survival upon osmotic downshifts. The data strongly suggest that L. lactis uses MscL as the main mechanosensitive solute release system to protect the cells under conditions of osmotic downshift.