ATP stimulates chemically sensitive and sensitizes mechanically sensitive afferents

We examined whether ATP stimulation of P2X purinoceptors would raise blood pressure in decerebrate cats. Femoral arterial injection of the P2X receptor agonist alpha,beta-methylene ATP into the blood supply of the triceps surae muscle induced a dose-dependent increase in arterial blood pressure. The maximal increase in mean arterial pressure (MAP) evoked by 0.1, 0.2, and 0.5 mM alpha,beta-methylene ATP (0.5 ml/min injection rate) was 6.2 +/- 2.5, 22.5 +/- 4.4, and 35.2 +/- 3.9 mmHg, respectively. The P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (2 mM ia) attenuated the increase in MAP elicited by intra-arterial alpha,beta-methylene ATP (0.5 mM), whereas the P2Y receptor antagonist reactive blue 2 (2 mM ia) did not affect the MAP response to alpha,beta-methylene ATP. In a second group of experiments, we tested the hypothesis that ATP acting through P2X receptors would sensitize muscle afferents and, thereby, augment the blood pressure response to muscle stretch. Two kilograms of muscle stretch evoked a 26.5 +/- 4.3 mmHg increase in MAP. This MAP response was enhanced when 2 mM ATP or 0.1 mM alpha,beta-methylene ATP (0.5 ml/min) was arterially infused 10 min before muscle stretch. Furthermore, this effect of ATP on the pressor response to stretch was attenuated by 2 mM pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (P < 0.05) but not by the P1 purinoceptor antagonist 8-(p-sulfophenyl)-theophylline (2 mM). These data indicate that activation of ATP-sensitive P2X receptors evokes a skeletal muscle afferent-mediated pressor response and that ATP at relatively low doses enhances the muscle pressor response to stretch via engagement of P2X receptors.     

Spinal P2X receptor modulates reflex pressor response to activation of muscle afferents

Static contraction of skeletal muscle evokes increases in blood pressure and heart rate. Previous studies suggested that the dorsal horn of the spinal cord is the first synaptic site responsible for those cardiovascular responses. In this study, we examined the role of ATP-sensitive P2X receptors in the cardiovascular responses to contraction by microdialyzing the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) into the L7 level of the dorsal horn of nine anesthetized cats. Contraction was elicited by electrical stimulation of the L7 and S1 ventral roots. Blockade of P2X receptor attenuated the contraction induced-pressor response [change in mean arterial pressure (delta MAP): 16 +/- 4 mmHg after 10 mM PPADS vs. 42 +/- 8 mmHg in control; P < 0.05]. In addition, the pressor response to muscle stretch was also blunted by PPADS (delta MAP: 27 +/- 5 mmHg after PPADS vs. 49 +/- 8 mmHg in control; P < 0.05). Finally, activation of P2X receptor by microdialyzing 0.5 mM alpha,beta-methylene into the dorsal horn significantly augmented the pressor response to contraction. This effect was antagonized by prior PPADS dialysis. These data demonstrate that blockade of P2X receptors in the dorsal horn attenuates the pressor response to activation of muscle afferents and that stimulation of P2X receptors enhances the reflex response, indicating that P2X receptors play a role in mediating the muscle pressor reflex at the first synaptic site of this reflex.     

Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response.

Activation of purinergic P2X receptors and transient receptor potential vanilloid type 1 (TRPV1) on muscle afferent nerve evokes the pressor response. Because P2X and TRPV1 receptors are sensitive to changes in pH, the aim of this study was to examine the effects of muscle acidification on those receptor-mediated cardiovascular responses. In decerebrate rats, the pH in the hindlimb muscle was adjusted by infusing acidic Ringer solutions into the femoral artery. Dialysate was then collected using microdialysis probes inserted into the muscles, and pH was measured. The interstitial pH was 7.53+/-0.01, 7.22+/-0.02, 6.94+/-0.04, and 6.59+/-0.03 in response to arterial infusion of the Ringer solution at pH 7.4, 6.5, 5.5, and 4.5, respectively. Femoral arterial injection of alpha,beta-methylene-ATP (P2X receptor agonist) in the concentration of 0.25 mM (volume, 0.15-0.25 ml; injection duration, 1 min) at the infused pH of 7.4, 6.5, and 5.5 increased mean arterial pressure (MAP) by 29+/-2, 24+/-3, and 21+/-3 mmHg, respectively (P<0.05, pH 5.5 vs. pH 7.4). When pH levels in the infused solution were 7.4, 6.5, 5.5, and 4.5, capsaicin (1 microg/kg), a TRPV1 agonist, was injected into the artery. This elevated MAP by 29+/-4, 33+/-2, 35+/-3, and 40+/-3 mmHg, respectively (P<0.05, pH 4.5 vs. pH 7.4). Furthermore, blocking acid-sensing ion channel (ASIC) blunted pH effects on TRPV1 response. Our data indicate that 1) muscle acidosis attenuates P2X-mediated pressor response but enhances TRPV1 response; 2) exaggerated TRPV1 response may require lower pH in muscle, and the effect is likely to be mediated via ASIC mechanisms. This study provides evidence that muscle pH may be important in modulating P2X and TRPV1 responsiveness in exercising muscle.     

Role played by P2X and P2Y receptors in evoking the muscle chemoreflex.

The role played by purinergic 2Y receptors in evoking the muscle chemoreflex is not well defined. To shed light on this issue, we compared the pressor responses with popliteal arterial injection of UTP (1 mg/kg), a selective P2Y agonist, with those to popliteal arterial injection of ATP (1 mg/kg), a P2X and P2Y agonist, and to alpha,beta-methylene ATP (50 mug/kg), a selective P2X1 and P2X3 agonist, in decerebrate unanesthetized cats. We found that injection of ATP and alpha,beta-methylene ATP increased mean arterial pressure by 19 +/- 2 and 15 +/- 4 mmHg, whereas UTP had no affect on arterial pressure. In addition, the pressor responses to injection of ATP and alpha,beta-methylene ATP were abolished by section of the sciatic nerve, demonstrating that they were reflex in origin. We conclude that P2Y receptors on thin fiber muscle afferents play no role in evoking the muscle chemoreflex.     

Exercise training prevents skeletal muscle afferent sensitization in rats with chronic heart failure

An exaggerated exercise pressor reflex (EPR) contributes to exercise intolerance and excessive sympathoexcitation in the chronic heart failure (CHF) state, which is prevented by exercise training (ExT) at an early stage in the development of CHF. We hypothesized that ExT has a beneficial effect on the exaggerated EPR by improving the dysfunction of muscle afferents in CHF. We recorded the discharge of mechanically sensitive (group III) and metabolically sensitive (group IV) afferents in response to static contraction, passive stretch, and hindlimb intra-arterial injection of capsaicin in sham+sedentary (Sed), sham+ExT, CHF+Sed, and CHF+ExT rats. Compared with sham+Sed rats, CHF+Sed rats exhibited greater responses of group III afferents to contraction and stretch, whereas the responses of group IV afferents to contraction and capsaicin were blunted. ExT prevented the sensitization of group III responses to contraction or stretch and partially prevented the blunted group IV responses to contraction or capsaicin in CHF rats. Furthermore, we investigated whether purinergic 2X (P2X) and transient receptor potential vanilloid 1 (TRPV1) receptors mediate the altered sensitivity of muscle afferents by ExT in CHF. We found that the upregulated P2X and downregulated TRPV1 receptors in L4/5 dorsal root ganglia of CHF rats were normalized by ExT. Hindlimb intra-arterial infusion of a P2X antagonist attenuated the group III response to contraction or stretch in CHF rats to a greater extent than in sham rats, which was normalized by ExT. These findings suggest that ExT improves the abnormal sensitization of muscle afferents in CHF at least, in part, via restoring the dysfunction of P2X and TRPV1 receptors.     

Activation of thin-fiber muscle afferents by a P2X agonist in cats.

The responses of group III and IV triceps surae muscle afferents to intra-arterial injection of alpha,beta-methylene ATP (50 microg/kg) was examined in decerebrate cats. We found that this P2X(3) agonist stimulated only three of 18 group III afferents but 7 of 9 group IV afferents (P < 0.004). The three group III afferents stimulated by alpha,beta-methylene ATP conducted impulses below 4 m/s. Pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid, a P2-receptor antagonist, prevented the stimulation of these afferents by alpha,beta-methylene ATP. We conclude that P2X(3) agonists stimulate only the slowest conducting group III muscle afferents as well as group IV afferents.     

Differential role of ionotropic glutamatergic mechanisms in responses to NTS P(2x) and A(2a) receptor stimulation

Activation of ATP P(2x) receptors in the subpostremal nucleus tractus solitarii (NTS) via microinjection of alpha,beta-methylene ATP (alpha,beta-MeATP) elicits fast initial depressor and sympathoinhibitory responses that are followed by slow, long-lasting inhibitory effects. Activation of NTS adenosine A(2a) receptors via microinjection of CGS-21680 elicits slow, long-lasting decreases in arterial pressure and renal sympathetic nerve activity (RSNA) and an increase in preganglionic adrenal sympathetic nerve activity (pre-ASNA). Both P(2x) and A(2a) receptors may operate via modulation of glutamate release from central neurons. We investigated whether intact glutamatergic transmission is necessary to mediate the responses to NTS P(2x) and A(2a) receptor stimulation. The hemodynamic and neural (RSNA and pre-ASNA) responses to microinjections of alpha,beta-MeATP (25 pmol/50 nl) and CGS-21680 (20 pmol/50 nl) were compared before and after pretreatment with kynurenate sodium (KYN; 4.4 nmol/100 nl) in chloralose-urethan-anesthetized male Sprague-Dawley rats. KYN virtually abolished the fast responses to alpha,beta-MeATP and tended to enhance the slow component of the neural responses. The depressor responses to CGS-21680 were mostly preserved after pretreatment with KYN, although the increase in pre-ASNA was reduced by one-half following the glutamatergic blockade. We conclude that the fast responses to stimulation of NTS P(2x) receptors are mediated via glutamatergic ionotropic mechanisms, whereas the slow responses to stimulation of NTS P(2x) and A(2a) receptors are mediated mostly via other neuromodulatory mechanisms.     

Comparative responses to alpha,beta-methylene-ATP in cat pulmonary, mesenteric, and hindquarter vascular beds.

Responses to the P2X-purinoceptor agonist alpha,beta-methylene-ATP (alpha,beta-MeATP) were investigated in the pulmonary, hindquarter, and mesenteric vascular beds in the cat. Under constant-flow conditions, injections of alpha,beta-MeATP caused dose-related increases in perfusion pressure in the pulmonary and hindquarter beds and a biphasic response in the mesenteric circulation. In the pulmonary vascular bed, the order of potency was alpha,beta-MeATP > U-46619 > angiotensin II, whereas, in the hindquarters, the order of potency was angiotensin II > U-46619 > alpha,beta-MeATP. The order of potency was similar in the hindquarter and mesenteric beds when the pressor component of the response to alpha,beta-MeATP was compared with responses to angiotensin II and U-46619. The P2X-receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid attenuated the pressor response to alpha,beta-MeATP in the hindquarter circulation and the pressor component in the mesenteric vascular bed. Pressor responses to alpha,beta-MeATP were not altered by cyclooxygenase, alpha-adrenergic, or angiotensin AT(1) antagonists. These data show that alpha,beta-MeATP has potent pressor activity in the pulmonary circulation, where it was 100-fold more potent than angiotensin II. In contrast, alpha,beta-MeATP had modest pressor activity in the systemic bed, where it was 1,000-fold less potent than angiotensin II. These data suggest that responses to alpha,beta-MeATP are dependent on the vascular bed studied and may be dependent on the density of P2X receptors in the vascular bed.     

P2X receptor-mediated muscle pressor reflex in myocardial infarction

A previous report from this laboratory demonstrated that the ATP-sensitive P2X receptor-mediated muscle pressor reflex was augmented in rats with heart failure (HF). The purpose of this study was to better understand the underlying mechanisms for this greater response in HF rats. We examined 1) responsiveness of the P2X receptor to alpha,beta-methylene ATP (alpha,beta-me-ATP), a P2X receptor agonist, in control and HF rats induced by myocardial infarction (MI); 2) the relationship between P2X-induced blood pressure response and left ventricular (LV) function; and 3) the expression of P2X receptors in the dorsal root ganglion (DRG) of control rats and rats with HF. Eight to 14 wk after coronary artery ligation, the severity of the MI was determined by echocardiography. In the first group of the experiment, alpha,beta-me-ATP (0.0625, 0.125, 0.25, and 0.5 mM) was injected into the arterial blood supply of the hindlimb muscles to evoke a pressor response in 17 decerebrated rats (6 controls, 6 small MIs with infarcts of the LV between 10 and 35%, and 5 large MIs with infarcts >35%). The P2X agonist increased blood pressure, and the effect was significantly accentuated in large MI rats compared with small MI rats and control rats. A significant correlation was observed between alpha,beta-me-ATP-evoked pressor response and the LV fractional shortening, an index of LV function. In the second group of the experiment, immunocytochemistry was used to examine the immunoreactivity of P2X receptor in the DRG neurons of small diameter fibers in six healthy control rats, five small MI, and five large MI rats. The percentage of P2X immunostaining-positive neurons in the DRG was markedly greater in large MI rats (52% vs. 29% in controls and 34% in small MIs, P < 0.05). In conclusion, our findings demonstrate that 1) muscle afferent-mediated pressor response of P2X activation was exaggerated in MI animals, and the responsiveness was related to the degree of LV dysfunction; and 2) augmented reflex response was associated with upregulated P2X receptors in the DRG neurons of thin fiber afferent nerves following MI. The data suggest that P2X-mediated responsiveness in the processing of muscle afferent signals may have important implications for understanding cardiovascular responses to exercise in HF.     

Local prostaglandin blockade attenuates muscle mechanoreflex-mediated renal vasoconstriction during muscle stretch in humans

During exercise, muscle mechanoreflex-mediated sympathoexcitation evokes renal vasoconstriction. Animal studies suggest that prostaglandins generated within the contracting muscle sensitize muscle mechanoreflexes. Thus we hypothesized that local prostaglandin blockade would attenuate renal vasoconstriction during ischemic muscle stretch. Eleven healthy subjects performed static handgrip before and after local prostaglandin blockade (6 mg ketorolac tromethamine infused into the exercising forearm) via Bier block. Renal blood flow velocity (RBV; Duplex Ultrasound), mean arterial pressure (MAP; Finapres), and heart rate (HR; ECG) were obtained during handgrip, post-handgrip muscle ischemia (PHGMI) followed by PHGMI with passive forearm muscle stretch (PHGMI + stretch). Renal vascular resistance (RVR, calculated as MAP/RBV) was increased from baseline during all paradigms except during PHGMI + stretch after the ketorolac Bier block trial where RVR did not change from baseline. Before Bier block, RVR rose more during PHGMI + stretch than during PHGMI alone (P < .01). Similar results were found after a saline Bier block trial (Delta53 +/- 13% vs. Delta35 +/- 10%; P < 0.01). However, after ketorolac Bier block, RVR was not greater during PHGMI + stretch than during PHGMI alone [Delta39 +/- 8% vs. Delta40 +/- 12%; P = not significant (NS)]. HR and MAP responses were similar during PHGMI and PHGMI + stretch (P = NS). Passive muscle stretch during ischemia augments renal vasoconstriction, suggesting that ischemia sensitizes mechanically sensitive afferents. Inhibition of prostaglandin synthesis eliminates this mechanoreceptor sensitization-mediated constrictor responses. Thus mechanoreceptor sensitization in humans is linked to the production of prostaglandins.     

P2X1 receptors and the endothelium

Adenosine triphosphate (ATP) is now established as a principle vaso-active mediator in the vasculature. Its actions on arteries are complex, and are mediated by the P2X and P2Y receptor families. It is generally accepted that ATP induces a bi-phasic response in arteries, inducing contraction via the P2X and P2Y receptors on the smooth muscle cells, and vasodilation via the actions of P2Y receptors located on the endothelium. However, a number of recent studies have placed P2X1 receptors on the endothelium of some arteries. The use of a specific P2X1 receptor ligand, alpha, beta methylene ATP has demonstrated that P2X1 receptors also have a bi-functional role. The actions of ATP on P2X1 receptors is therefore dependant on its location, inducing contraction when located on the smooth muscle cells, and dilation when expressed on the endothelium, comparable to that of P2Y receptors.     

Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens.

Membranes of the rat vas deferens were shown to contain a high density of binding sites for [3H] alpha, beta-methylene ATP ([3H] alpha, beta-MeATP), a ligand selective for the P2X purinoceptor. Analysis demonstrated two classes, of high affinity (Kd = 1.8 nM, Bmax (maximum density) = 9.3 pmol/mg of protein) and of low affinity (Kd = 34 nM, Bmax = 29 pmol/mg of protein). The high affinity [3H] alpha, beta-MeATP binding sites were successfully solubilized with 2% digitonin: the Kd was then 1.6 nM. Both the association and dissociation of the receptor-ligand complex were rapid (half-time for association = 6.5 min). The rank order of potency of purinergic ligands in displacing [3H] alpha, beta-MeATP binding from the solubilized preparation was in accord with the pharmacological criteria for P2X purinoceptors. The receptor-detergent complex was separated by sucrose gradient ultracentrifugation from the ATPase enzymes also present in the preparation. The sedimentation coefficient of the receptor-detergent complex was 12.1 S. It was shown that [3H] alpha, beta-MeATP can function as a photoaffinity labeling reagent upon exposure to ultraviolet light; in the rat vas deferens membranes, it thus became cross-linked in a specific manner to a polypeptide of apparent molecular mass = 62,000 daltons, proposed to be the ligand-binding subunit of the functional P2X purinoceptor.     

alpha,beta-Methylene ATP elicits a reflex pressor response arising from muscle in decerebrate cats.

In part, the exercise pressor reflex is believed to be evoked by chemical stimuli signaling that blood supply to exercising muscles is not adequate to meet its metabolic demands. There is evidence that either ATP or adenosine may function as one of these chemical stimuli. For example, muscle interstitial concentrations of both substances have been found to increase during exercise. This finding led us to test the hypothesis that popliteal arterial injection of alpha,beta-methylene ATP (5, 20, and 50 microg/kg), which stimulates P2X receptors, and 2-chloroadenosine (25 microg/kg), which stimulates P1 receptors, evokes reflex pressor responses in decerebrate, unanesthetized cats. We found that popliteal arterial injection of the two highest doses of alpha,beta-methylene ATP evoked pressor responses, whereas popliteal arterial injection of 2-chloroadenosine did not. In addition, the pressor responses evoked by alpha,beta-methylene ATP were blocked either by section of the sciatic nerve or by prior popliteal arterial injection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (10 mg/kg), a selective P2-receptor antagonist. We conclude that the stimulation of P2 receptors, which are accessible through the vascular supply of skeletal muscle, evokes reflex pressor responses. In addition, our findings are consistent with the hypothesis that the stimulation of P2 receptors comprises part of the metabolic error signal evoking the exercise pressor reflex.     

Role played by purinergic receptors on muscle afferents in evoking the exercise pressor reflex.

The exercise pressor reflex is believed to be evoked, in part, by multiple metabolic stimuli that are generated when blood supply to exercising muscles is inadequate to meet metabolic demand. Recently, ATP, which is a P2 receptor agonist, has been suggested to be one of the metabolic stimuli evoking this reflex. We therefore tested the hypothesis that blockade of P2 receptors within contracting skeletal muscle attenuated the exercise pressor reflex in decerebrate cats. We found that popliteal arterial injection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 10 mg/kg), a P2 receptor antagonist, attenuated the pressor response to static contraction of the triceps surae muscles. Specifically, the pressor response to contraction before PPADS averaged 36 +/- 3 mmHg, whereas afterward it averaged 14 +/- 3 mmHg (P < 0.001; n = 19). In addition, PPADS attenuated the pressor response to postcontraction circulatory occlusion (P < 0.01; n = 11). In contrast, popliteal arterial injection of CGS-15943 (250 micro g/kg), a P1 receptor antagonist, had no effect on the pressor response to static contraction of the triceps surae muscles. In addition, popliteal arterial injection of PPADS but not CGS-15943 attenuated the pressor response to stretch of the calcaneal (Achilles) tendon. We conclude that P2 receptors on the endings of thin fiber muscle afferents play a role in evoking both the metabolic and mechanoreceptor components of the exercise pressor reflex.     

ATPalphaS is a ligand for P2Y receptors in synaptosomal membranes: solubilization of [35S]ATPalphaS binding proteins associated with G-proteins.

ATPalphaS was established as a P2Y receptor-specific ligand for assaying the solubilization of functional native P2Y receptors from synaptosomal membranes. These receptors are not yet amenable to biochemical studies. High-affinity [35S]ATPalphaS binding sites in synaptosomal membranes, solubilized with Brij58, retained the binding affinity and ligand specificity (ATPalphaS = ATP > 2-MeSATP > ADP, ADPbetaS > AMP > alpha,beta-MeATP) corresponding to P2Y receptors. Mg2+ but not Ca2+, enhanced high-affinity [35S]ATPalphaS binding 30-fold, supporting specific recognition by P2Y receptors. ATPalphaS stimulated P2Y receptor-mediated [35S]GTPgammaS binding equipotently with ATP in synaptosomal membranes and in Brij58-solubilized proteins demonstrating the association with G-proteins. Anion-exchange chromatography of solubilized synaptosomal membrane proteins yielded two fractions in which [35S]ATPalphaS binding was regulated by GTPgammaS/Mg2+, thus possibly by heterotrimeric G-proteins. After a second chromatographic step (hydroxyapatite) the regulation of high-affinity [35S]ATPalphaS binding by Mg2+ was still present, whereas the regulation by GTPgammaS/Mg2+ was lost indicating the dissociation from G-proteins. Thus, conditions were found to stabilize ligand binding activity of solubilized P2Y receptors and to solubilize P2Y receptors associated with G-proteins.     

Blockade of purinergic 2 receptors attenuates the mechanoreceptor component of the exercise pressor reflex

The finding that pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), a P2 antagonist, attenuated the pressor response to calcaneal tendon stretch, a purely mechanical stimulus, raises the possibility that P2 receptors sensitize mechanoreceptors to static contraction of the triceps surae muscles. The mechanical component of the exercise pressor reflex, which is evoked by static contraction, can be assessed by measuring renal sympathetic nerve activity during the first 2-5 s of this maneuver. During this period of time, group III mechanoreceptors often discharge explosively in response to the sudden tension developed at the onset of contraction. In decerebrated cats, we, therefore, examined the effect of PPADS (10 mg/kg) injected into the popliteal artery on the renal sympathetic and pressor responses to contraction and stretch. We found that PPADS significantly attenuated the renal sympathetic response to contraction, with the effect starting 2 s after its onset and continuing throughout its 60-s period. PPADS also significantly attenuated the renal sympathetic nerve response to stretch, but did so after a latency of 10 s. Our findings lead us to conclude that P2 receptors sensitize group III muscle afferents to contraction. The difference in the onset latency between the PPADS-induced attenuation of the renal sympathetic response to contraction and the renal sympathetic response to stretch is probably due to the sensitivities of different populations of group III afferents to ATP released during contraction and stretch.     

Trehalose uptake through P2X7 purinergic channels provides dehydration protection

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.     

Identification of P1 and P2 purinoceptors in the aorta of the lizard (Agama sp.)

In the isolated Agama lizard aorta, acetylcholine (ACh; 3 nM-100 microM), noradrenaline (NA; 30 nM-0.3 mM), adrenaline (Adr; 30 nM-300 microM), adenosine 5'-triphosphate (ATP; 30 nM-1 mM), alpha,beta-methylene ATP (alpha,beta-meATP; 10 nM-10 microM), beta,gamma-methylene ATP (beta,gamma-meATP; 0.1-300 microM), 2-methylthio ATP (2-meSATP; 30 nM-30 microM) and high concentrations of uridine triphosphate (UTP; 1 microM-1 mM), all produced constriction. The P2 receptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; 30 microM), suramin (0.1 mM) and Reactive blue 2 (30 microM) all raised vascular tone and could not be utilized and the antagonist 2'-O-(trinitrophenyl) ATP (TNP-ATP; 0.1 microM) had no effect on responses to the ATP analogues. alpha,beta-MeATP (3 microMx3) desensitised responses to alpha,beta-meATP (10 microM) and beta,gamma-meATP (0.3 mM), but not to ATP (0.3 mM) or 2-meSATP (30 microM). On pre-constricted aorta (EC50 concentration of either ACh or Adr), adenosine (1 microM-1 mM), the A1-selective agonist N6-cyclopentyl adenosine (CPA; 1-300 microM) [but not the A2- and A3-selective agonists CGS 21680 and IB-MECA respectively (both up to 30 microM)] and sodium nitroprusside (10 nM-100 microM) produced vasodilatation. Adenosine vasodilatation was antagonised by 8-p-sulfophenyl-theophylline (8-pSPT; 30 microM) but not by N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.1 mM). ATP (up to 0.3 mM), 2-meSATP (up to 10 microM) and UTP (up to 1 mM) were not vasodilators. In summary, A1 receptors mediating relaxation and excitatory P2X1 receptors were identified in the smooth muscle of the lizard aorta. However, in contrast to mammalian aorta, P2Y receptors on endothelial cells mediating vasodilatation via nitric oxide do not appear to be present.