The Joan Mott Prize Lecture. The integrated response to hypoxia: from circulation to cells.
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Adenosine↗
Biomedical subjects
J M Marshall
Publications and source records attributed to J M Marshall.
Evidence that the conformation of unliganded human plasminogen is maintained via an intramolecular interaction between the lysine-binding site of kringle 5 and the N-terminal peptide.
Human Glu-plasminogen adopts at least three conformations that provide a means for regulating the specificity of its activation in vivo. It has been proposed previously that the closed (alpha) conformation of human Glu-plasminogen is maintained through physical interaction of the kringle 5 domain and a lysine residue within the N-terminal peptide (NTP). To examine this hypothesis, site-directed mutagenesis was used to generate variant proteins containing substitutions either for aspartic acid residues within the anionic centre of the kringle 5 domain or for conserved lysine residues within the NTP. Size-exclusion HPLC and rates of plasminogen activation by urokinase-type plasminogen activator were used to determine the conformational states of these variants. Variants with substitutions within the kringle 5 lysine-binding site demonstrated extended conformations, as did variants with alanine substitutions for Lys50 and Lys62. In contrast, molecules in which NTP residues Lys20 or Lys33 were replaced were shown to adopt closed conformations. We conclude that the lysine-binding site of kringle 5 is involved in maintaining the closed conformation of human Glu-plasminogen via an interaction with the NTP, probably through Lys50 and/or Lys62. These conclusions advance the current model for the initial stages of fibrinolysis during which fibrin is thought to compete with the NTP for the kringle 5 lysine-binding site.
Amino Acid Substitution↗
Role of adenosine and its receptors in the vasodilatation induced in the cerebral cortex of the rat by systemic hypoxia.
1. In anaesthetized rats, we have examined the role of adenosine in vasodilatation evoked in the cerebral cortex by systemic hypoxia (breathing 8 % O2). Red cell flux was recorded from the surface of the exposed parietal cortex (CoRCF) by a laser Doppler probe, cortical vascular conductance (CoVC) being computed as CoRCF divided by mean arterial blood pressure. All agonists and antagonists were applied topically to the cortex. 2. Systemic hypoxia or adenosine application for 5 or 10 min, respectively, induced an increase in CoRCF and CoVC. These responses were substantially reduced by 8-phenyltheophylline (8-PT), an adenosine receptor antagonist which is non-selective between the adenosine A1 and A2A receptor subtypes. By contrast, the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT) which is similarly non-selective, but unlike 8-PT, does not cross the blood-brain barrier, reduced the increases in CoRCF and CoVC induced by adenosine, but had no effect on those induced by hypoxia. 3. The A2A receptor agonist CGS21680 produced a substantial increase in CoRCF and CoVC, but the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine had minimal effects. 4. The A2A receptor antagonist ZM241385 reduced the increase in CoRCF and CoVC induced by adenosine and reduced the increase in CoRCF induced by hypoxia. 5. We propose that exogenous adenosine that is topically applied to the cerebral cortex produces vasodilatation by acting on A2A receptors on the vascular smooth muscle. However, during systemic hypoxia, we propose that adenosine is released from endothelial cells and acts on endothelial A2A receptors to produce the major part of the hypoxia-induced dilatation in the cerebral cortex.
Adenosine↗
Comparison of responses evoked by mild indirect cooling and by sound in the forearm vasculature in patients with homozygous sickle cell disease and in normal subjects.
In normal individuals, novel or noxious stimuli commonly evoke the pattern of the alerting or defence response which includes cutaneous vasoconstriction, but vasodilatation in forearm skeletal muscle. We have compared cardiovascular responses evoked by sound and by indirect cooling in 60 patients with homozygous sickle cell (SS) disease and in 30 control subjects with normal haemoglobin genotype (AA). A sound of 90 dB, 1 kHz for 30s evoked an increase in hand and forearm cutaneous vascular resistance (HCVR and FCVR) in SS patients and an increase in HCVR in AA subjects, as assessed from Doppler flowmetry. Meanwhile, a decrease in forearm vascular resistance (FVR) assessed by venous occlusion plethysmography, occurred in 14 out of 30 AA subjects and 25 out of 60 SS patients, indicating vasodilatation in forearm muscle; an increase in FVR occurred in the remainder. The proportions of SS patients and AA subjects who showed an increase in FVR (53% vs 57%) were not significantly different. Cooling increased HCVR and FCVR in SS patients and increased FCVR in AA subjects; a decrease in FVR indicating vasodilatation, occurred in 12 out of 30 AA subjects, but in only 10 out of 60 SS patients. The proportion of SS patients who showed an increase in FVR to cooling was greater than in AA subjects (83% vs 60%, P < 0.05). Thus, SS patients are just as capable of showing the muscle vasodilatation of the alerting response to sound as AA subjects. That few SS patients showed muscle vasodilatation in response to cooling is consistent with the view that reflex vasoconstrictor responses to cooling are particularly strong in SS patients. This, in turn, is consistent with our hypothesis that the reflex vasoconstrictor response to cooling acts as a trigger for the painful crisis of SS disease by diverting blood flow away from active bone marrow.
Acoustic Stimulation↗
Peripheral vascular response to mild indirect cooling in patients with homozygous sickle cell (SS) disease and the frequency of painful crisis.
1. In homozygous sickle cell (SS) disease, skin cooling is a common precipitating factor of the painful crisis which is associated with avascular necrosis of active bone marrow. Since skin cooling does not directly induce sickling, we have investigated the nature of the reflex vascular responses to mild cooling in SS patients in a steady state of the disease and compared them with their history of painful crises. 2. Experiments were performed in Jamaica on 60 male SS patients and 30 matched control subjects with normal haemoglobin (AA) genotype. Forearm blood flow (FBF) was measured by venous occlusion plethysmography and mean arterial pressure (MAP) by a Finapres device: forearm vascular resistance (FVR) was calculated as MAP/FBF. Cutaneous erythrocyte flux in forearm and hand was monitored by a laser Doppler meter. The contralateral hand was immersed in cool water at 16 degrees C for 2 min, 6 times, at random intervals of 0.5-3 min. 3. The first cool immersion evoked an increase in MAP, cutaneous vasoconstriction and a net increase in FVR in both AA and SS subjects. However, the direction of change in FVR varied between individuals such that 18 AA subjects showed an increase in FVR (constrictor group) while 12 showed a decrease in FVR, indicating vasodilatation in forearm muscle (dilator group). In contrast, 50 SS subjects showed an increase in FVR and only 10 showed a decrease in FVR. The proportion of subjects who showed net vasoconstriction was significantly greater in the SS than in the AA group (83% versus 60%, P = 0.03, chi 2 test). 4. By the sixth cool stimulus, the 'dilator' group of AA subjects showed no change in FVR while the 'dilator' group of SS patients showed an increase in FVR. We suggest that forearm muscle vasodilatation was the characteristic component of the alerting/defence response to novel or noxious stimuli which habituates on repetition. 5. In the whole group of SS patients, baseline values of cutaneous vascular resistance and FVR increased between stimuli, indicating persistent vasoconstriction, and the sixth cool stimulus still evoked cutaneous vasoconstriction and a net increase in FVR. In contrast, AA subjects showed an increase in baseline FVR between stimuli, but the sixth cool stimulus had no significant effect on cutaneous vascular resistances, or FVR. 6. In SS patients there were no associations between the direction of change in FVR evoked by the first cool stimulus and forearm circumference or skinfold thickness, concentrations of haemoglobin or fetal haemoglobin. However, the frequency of painful crises was significantly greater in the 'constrictor' group than in the 'dilator' group (0.36 versus 0.12/year, P = 0.04, Mann-Whitney test). 7. These results indicate that the primary reflex vasoconstrictor response evoked by mild cooling is stronger and more persistent in SS patients than in AA subjects and is particularly strong in SS patients who are most prone to painful crises. The results are consistent with the hypothesis that skin cooling may precipitate the painful crisis by causing reflex vasoconstriction in muscle, and possibly in bone marrow, so diverting blood flow away from the active marrow.
Acute Disease↗
Chemoreceptors and cardiovascular control in acute and chronic systemic hypoxia.
This review describes the ways in which the primary bradycardia and peripheral vasoconstriction evoked by selective stimulation of peripheral chemoreceptors can be modified by the secondary effects of a chemoreceptor-induced increase in ventilation. The evidence that strong stimulation of peripheral chemoreceptors can evoke the behavioural and cardiovascular components of the alerting or defence response which is characteristically evoked by novel or noxious stimuli is considered. The functional significance of all these influences in systemic hypoxia is then discussed with emphasis on the fact that these reflex changes can be overcome by the local effects of hypoxia: central neural hypoxia depresses ventilation, hypoxia acting on the heart causes bradycardia and local hypoxia of skeletal muscle and brain induces vasodilatation. Further, it is proposed that these local influences can become interdependent, so generating a positive feedback loop that may explain sudden infant death syndrome (SIDS). It is also argued that a major contributor to these local influences is adenosine. The role of adenosine in determining the distribution of O2 in skeletal muscle microcirculation in hypoxia is discussed, together with its possible cellular mechanisms of action. Finally, evidence is presented that in chronic systemic hypoxia, the reflex vasoconstrictor influences of the sympathetic nervous system are reduced and/or the local dilator influences of hypoxia are enhanced. In vitro and in vivo findings suggest this is partly explained by upregulation of nitric oxide (NO) synthesis by the vascular endothelium which facilitates vasodilatation induced by adenosine and other NO-dependent dilators and attenuates noradrenaline-evoked vasoconstriction.
Acute Disease↗
Lack of habituation of the pattern of cardiovascular response evoked by sound in subjects with primary Raynaud's disease.
1. The vasospasm of primary Raynaud's disease can be triggered by acute emotional stress. We have studied the pattern of cardiovascular response evoked by acute emotional stress, a sound stimulus of 90 dB, 2 kHz for 30 s, in eight subjects with primary Raynaud's disease and in eight age- and sex-matched controls, the sound being repeated five times on each of days 1, 3 and 5. 2. In controls, the first sound evoked the pattern of the alerting response that is characteristic of acute emotional stress: a rise in arterial pressure and heart rate, a decrease in vascular conductance in the cutaneous circulation of the digit, assessed by laser Doppler recording of erythrocyte (red cell) flux in the digit divided by arterial pressure, and an increase in forearm muscle vascular conductance, assessed from forearm blood flow recorded by venous occlusion plethysmography divided by arterial pressure. 3. In the subjects with primary Raynaud's disease, baselines of arterial pressure, digital cutaneous vascular conductance and forearm vascular conductance were not significantly different from those of the controls and they too showed the alerting response to the first sound, the magnitudes of the changes being comparable to those of the controls. 4. In both the controls and subjects with primary Raynaud's disease, the evoked responses were consistent on repetition of the sound on day 1. In contrast, judging from the means of the changes evoked on each day, the controls showed habituation of the individual components of the alerting response over days 1, 3 and 5, whereas the subjects with primary Raynaud's disease showed no habituation of either the forearm muscle vasodilatation or the digital vasoconstriction. Conversely, the decrease in digital cutaneous vascular conductance evoked by a single deep breath was fully reproducible in both controls and subjects with primary Raynaud's disease when tested at the beginning and end of each experimental day. 5. These results allow the novel conclusion that subjects with primary Raynaud's disease have an abnormality of the central neural modulation of the brain stem areas that integrate the cardiovascular components of the alerting response to acute emotional stress, such that habituation of the vasodilator and vasoconstrictor components of the response on repetition of the stimulus is impaired. We propose that such persistence of vasoconstrictor responses to stressful stimuli predisposes to vasospasm, particularly if neurally mediated vasoconstriction is reinforced by locally released vasoconstrictor factors.
Adult↗
Practical, ongoing competency-assessment program for hospital pharmacists and technicians.
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Attitude of Health Personnel↗
The roles of adenosine in regulating the respiratory and cardiovascular systems in chronically hypoxic, adult rats.
1. We have investigated the roles of adenosine in regulating the respiratory and cardiovascular systems of rats that were made chronically hypoxic for 3-4 weeks from 6 weeks of age (CH rats) in an hypoxic chamber at 12% O2. They were studied under anaesthesia while breathing 12% O2 and during acute hypoxia (breathing 8% O2 for 5 min) before and after addition of the adenosine receptor antagonist 8-phenyltheophylline (8-PT, 10 mg kg-1). The results were compared with those obtained from normoxic (N) rats in a previous study. 2. CH rats breathing 12% O2 had greater minute ventilation (VP) than N rats breathing air, but their levels of arterial blood pressure (ABP), heart rate (HR), femoral vascular conductance (FVC) and cerebral vascular conductance (CVC) were fully comparable. 8-PT increased tidal volume (VT) in CH rats indicating a greater tonic central inhibitory influence of adenosine on VT than in N rats. However, 8-PT had no effect on cardiovascular variables, indicating no tonic cardioinhibitory or vasodilator influence of adenosine in CH rats. 3. Acute hypoxia in CH rats increased VE such that at the 5th minute of 8% O2 absolute VE was comparable to that of N rats breathing 8% O2. Moreover, in CH rats 8-PT increased VT at the 5th minute of 8% O2 indicating that the central inhibitory influence of adenosine limits the ability to maintain VT in acute hypoxia as it does in N rats. 4. Eight per cent O2 also produced a full in ABP in CH rats that was comparable to that induced in N rats by the larger change from air to 8% O2. However, the changes in HR were similar in CH and N rats while the increases in FVC and CVC were smaller in CH rats. This suggests that the ability of the secondary effects of hyperventilation and of the baroreceptor reflex to maintain cardiac output and thereby ABP is reduced in CH rats. 5. Whereas 8-PT substantially reduced the hypoxia-induced increases in FVC and CVC in N rats, it had a small effect in CH rats (P = 0.054 and 0.06, respectively). Further, acute hypoxia in CH rats had no effect on the K+ concentration in the venous efflux of hindlimb K+ (KV+) before or after 8-PT treatment. We suggest that in CH rats, the dilator influence of adenosine in acute hypoxia occurs via actions on the blood vessel walls: there was no evidence that adenosine can release dilator concentrations of K+ from skeletal muscle fibres in CH rats as proposed for N rats.
Adenosine↗
The effects of chronic hypoxia on renal function in the rat.
1. Studies were performed on rats that had been made chronically hypoxic (CH rats) in a normoxic chamber at 12% O2 for 3-5 weeks. Under Saffan anaesthesia, respiratory and cardiovascular variables, renal haemodynamics and renal function were recorded while the rats spontaneously breathed 12% O2 followed by a switch to air breathing for 20 min. Plasma renin activity was assessed by radioimmunoassay of angiotensin I. Plasma atrial natiruetic peptide (ANP) was indirectly assessed by measurement of cyclic GMP in urine. 2. When breathing 12% O2, CH rats showed hyperventilation and raised haematocrit (52%) relative to normoxic (N) rats. But arterial pressure (ABP), renal blood flow (RBF), renal vascular conductance (RVC), mean right atrial pressure (mRAtP), urine flow, glomerular filtration rate (GFR) and absolute sodium excretion (UNaV) were comparable to those recorded in N rats breathing air. Urinary cGMP was 40% greater than in N rats, but plasma renin activity was not significantly greater in CH than in N rats. 3. Air breathing in CH rats induced hypoventilation, a 12% increase in ABP, no change in mRAtP, RBF or GFR, but increases of 75 and 100% in urine flow and UNaV, respectively. Neither urinary cGMP nor plasma renin activity changed. Such increases in urine flow and UNaV were absent when renal perfusion pressure (RPP) was prevented from rising during air breathing by using an occluder on the dorsal aorta. 4. We propose that by 3-5 weeks of chronic hypoxia renal function was normalized, principally because arterial O2 content was normalized by the increase in haematocrit and because ABP and renal haemodynamics were normalized: acute hypoxia in N rats produces a fall in ABP. We suggest that plasma ANP was raised by the actions of hypoxia or erythropoietin on the atrium, rather than by atrial distension, but suggest that ANP had little direct influence on renal function and tended to limit the influence of the renin-angiotensin system. We further propose that the diuresis and natriuresis seen during air breathing were mediated by the increase in RPP; neither plasma ANP nor renin activity change in the immediate short term.
Animals↗
Postural vasoconstriction and leg ulceration in homozygous sickle cell disease.
1. Chronic leg ulceration is a major cause of morbidity in patients with homozygous sickle cell disease; the ulcers commonly resolve on bed rest. We have therefore compared the cutaneous vascular response to dependency in three groups of eight patients with sickle cell disease (those with an active ulcer, with an ulcer scar and with no history of ulceration) and in eight subjects with normal haemoglobin and no history of leg ulceration. 2. We monitored, with a laser Doppler flowmeter, the change in red cell (erythrocyte) flux induced in the skin of the leg, at two sites proximal to the malleoli, with the leg horizontal and 5 and 10 min after moving the leg to the dependent position. 3. With the leg horizontal, mean cutaneous red cell flux was substantially higher in normal skin of patients with sickle cell disease than in normal subjects and was higher still at the site of the ulcer or scar. On dependency, red cell flux fell not only in normal subjects but also in the patients with sickle cell disease, both in the normal skin and at the site of the ulcer or scar; there was no difference in any group between the 5- and 10-min values. The fall in red cell flux in normal skin of patients with sickle cell disease was smaller than in normal subjects when considered as a percentage of the control values (32%, 36%, 30% and 61% respectively in sickle cell patients with an active ulcer, with an ulcer scar and with no history of ulceration and in normal subjects), but in absolute terms the falls in red cell flux were similar in sickle cell patients and normal subjects. By contrast, the fall in red cell flux at the ulcer or scar site was greater than in normal skin from sickle cell patients whether considered as a percentage of the control value (48% and 49% respectively in those with an active ulcer or ulcer scar) or in absolute terms. 4. We propose that high resting perfusion is important in patients with sickle cell disease to maintain normal integrity of cutaneous tissue and that pronounced vasoconstriction on dependency hinders the healing and encourages recurrence of leg ulcers.
Adult↗
Studies on the roles of ATP, adenosine and nitric oxide in mediating muscle vasodilatation induced in the rat by acute systemic hypoxia.
1. In Saffan-anaesthetized rats, we have further investigated the mechanisms underlying the vasodilatation induced by adenosine in skeletal muscle by acute systemic hypoxia (breathing 8% O2 for 5 min). 2. In eleven rats the nitric oxide (NO) synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 10 mg kg-1, i.v.) reduced the increase in femoral vascular conductance (FVC) induced by hypoxia by approximately 50%. L-NAME had similar effects on the increase in FVC induced by intra-arterial (I.A.) infusion of adenosine (at 1.2 mg kg-1 min-1 for 5 min via the tail artery) and by ATP (I.A., 1 mg kg-1 min-1 for 5 min). Subsequent administration of the adenosine receptor antagonist 8-sulphophenyl theophylline (8-SPT, 20 mg kg-1, i.v.) virtually abolished the adenosine- and ATP-induced increase in FVC. 3. In a further nine rats, 8-SPT reduced the increase in FVC induced by hypoxia by approximately 50%. This remaining increase in FVC was substantially reduced by L-NAME. 4. In an additional nine rats, alpha,beta-methyleneADP (160 micrograms kg-1, i.v.) which inhibits the 5'-ectonucleotidase that degrades AMP to adenosine, reduced the peripheral vasodilatation (fall in arterial blood pressure, ABP) induced by ATP infusion, but had no effect on the increase in FVC or decrease in ABP evoked by systemic hypoxia. 5. These results provide the first evidence that the muscle vasodilatation induced by adenosine during systemic hypoxia is mainly dependent on NO synthesis. They also suggest that adenosine is released as such rather than being formed extracellularly from AMP. Given evidence that extraluminal adenosine acts in an NO-independent fashion we propose that hypoxia releases adenosine from the endothelium. Our results also indicate that hypoxia induces muscle vasodilatation that is adenosine independent but NO dependent: they allow the possibility that this is partly mediated by ATP released from the endothelium.
5'-Nucleotidase↗
Postnatal development of the pattern of respiratory and cardiovascular response to systemic hypoxia in the piglet: the roles of adenosine.
1. In 3-day-old and 3-week-old spontaneously breathing piglets anaesthetized with Saffan, we have studied ventilatory and cardiovascular responses evoked by 5 min periods of hypoxia (breathing 10 and 6% O2). 2. In 3-day-old piglets both 10 and 6% O2 evoked an increase followed by a secondary fall in ventilation, a gradual tachycardia and a renal vasoconstriction, with an increase in femoral blood flow that was attributable to femoral vasodilatation. Arterial blood pressure rose initially but fell towards control values by the 5th minute of hypoxia. 3. The stable adenosine analogue 2-chloroadenosine (2-CA; 30 mg kg(-1) i.v.) evoked bradycardia and renal vasoconstriction, but had no effect on femoral vasculature. These responses were blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 8 mg kg(-1) i.v.). 8-PT also abolished the secondary fall in ventilation evoked by 10 and 6% O2 and the renal vasoconstriction evoked by 10% O2, but had no effect on the tachycardia, or on the femoral vascular response. 4. By contrast, in 3-week-old piglets both 10 and 6% O2 evoked a sustained increase in ventilation, tachycardia and a rise in arterial pressure with renal vasoconstriction, but no change in renal blood flow and substantial femoral vasodilatation with an increase in femoral blood flow. 2-CA evoked bradycardia and renal vasoconstriction, as in 3-day-old piglets, but also evoked pronounced femoral vasodilatation. 8-PT blocked these responses and the hypoxia-induced femoral vasodilatation, but had no significant effect on other components of the hypoxia-induced response. 5. We propose that there is postnatal development of the ventilatory and cardiovascular responses evoked by systemic hypoxia and of the role of locally released adenosine in these responses: at 3 days, adenosine released within the central nervous system and within the kidney is a major contributor to the secondary fall in ventilation and renal vasoconstriction respectively, whereas at 3 weeks, adenosine makes little contribution to the ventilatory response, or renal vasoconstriction, but is largely responsible for hypoxia-induced vaso-dilatation in skeletal muscle.
2-Chloroadenosine↗
The behaviour of muscle microcirculation in chronically hypoxic rats: the role of adenosine.
1. In rats housed in a hypoxic chamber at 12% O2 for 3-5 weeks (CH) and in normal rats housed in air (N), we directly observed responses of arterial and venous vessels of the spinotrapezius muscle to changes in O2 concentration in the inspirate. Both CH and N rats were anaesthetized with Saffan. They had haematocrits of 55.0 +/- 0.9% (mean +/- S.E.M.) and 41.9 +/- 0.5%, respectively. 2. In CH rats breathing 12% O2 and N rats breathing air, arterial and venous vessels from comparable anatomical positions in the vascular tree were of similar internal diameter. They also showed similar maximum dilator responses to topical adenosine (10(-3) M); 14.1 +/- 1.1 and 16.3 +/- 1.7% in all arterioles, 15.5 +/- 1.2 and 11.5 +/- 0.6% in all venules in CH and N rats, respectively. 3. In CH rats, the change from 12% O2 to air for 3 min induced constriction in all arterioles and venules (-12.9 +/- 1.0 and -14.3 +/- 1.7%, respectively), whereas in N rats, the change from air to 12% O2 for 3 min induced net dilatation (3.9 +/- 1.8% in arterioles and 4.7 +/- 0.8% in venules). Topical application of the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT, 10(-3) M) had no effect on control diameters in CH or N rats, nor on constrictor responses to air in CH, but reversed or reduced dilator responses to 12% O2 in N rats (to -2.4 +/- 1.3% in arterioles and 2.0 +/- 0.9% in venules). 4. In CH rats, the change from 12 to 8% O2 produced net dilatation as great as that induced in N rats by the larger change from air to 8% O2: 8.5 +/- 2.6 and 5.0 +/- 3.7% in arterioles and 10.3 +/- 1.8 and 6.4 +/- 1.9% in venules, respectively. These responses were similarly reduced by 8-SPT to -4.3 +/- 1.9 and -5.2 +/- 2.7% in arterioles and to -6.9 +/- 2.0 and -1.5 +/- 2.0% in venules, respectively. 5. These results indicate that CH rats were acclimated to 12% O2 such that the resting tone of arterial and venous vessels of muscle was comparable to that of N rats breathing air. They also suggest that adenosine had little tonic dilator influence in CH rats breathing 12% O2 despite its contribution to the dilatation induced in N rats by acute exposure to 12% O2. This may reflect the greater haematocrit in CH rats which normalized the O2 supply to muscle. However, CH rats were more sensitive than N rats to the dilator influence of acute systemic hypoxia and this was largely mediated by adenosine.
Adenosine↗
The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.
Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.
Binding Sites↗
The role of adenosine in mediating vasodilatation in mesenteric circulation of the rat in acute and chronic hypoxia.
1. We have compared the roles of adenosine in mediating dilator responses to acute hypoxia in mesenteric microcirculation of control, normoxic (N) rats and in chronically hypoxic (CH) rats kept in an hypoxic chamber at 10% O2 for 3-4 weeks. 2. In fifteen N rats, acute hypoxia (breathing 6% O2 for 3 min) induced mean increases in the diameter of arterial vessels of mesentery (whose internal diameter was 10-350 microns) of 8.0 +/- 1.9% (mean +/- S.E.M.) and of venous vessels (whose internal diameter was 12-360 microns) of 10.4 +/- 2.6%. These diameter changes were reduced by approximately 30% when the adenosine receptor antagonist 8-sulpho-phenyltheophylline (8-SPT, 10(-3) M) was applied topically to the mesentery. 3. In a further six N rats, topical application of graded concentrations of adenosine (10(-7)-10(-3) M) to the mesentery evoked graded increases in the diameter of all arterial and venous vessels, maximum increases with 10(-3) M being 12.5 +/- 3.3 and 8.4 +/- 4.3%, respectively; these responses were abolished by 8-SPT. 4. By contrast, in fourteen CH rats, the smaller change in inspirate from 10 to 8% O2 induced increases in diameter of arterial and venous vessels which had control diameters that were comparable to those of N rats, of 14.1 +/- 2.4 and 12.9 +/- 2.7%, respectively, and which were virtually equivalent to the responses induced by topical application of 10(-3) M adenosine (13.3 +/- 1.3 and 16.3 +/- 2.0% in arterial and venous vessels, respectively). The changes induced by acute hypoxia were abolished by 8-SPT, as were those induced by adenosine. 5. These results suggest that in the intestinal mesentery, where the blood vessels have negligible tissue parenchyma around them, locally released or synthesized adenosine makes a substantial contribution to the dilatation that is evoked in arteriolar vessels by acute hypoxia and to the active dilatation, or passive distension of the venous vessels. The results also suggest that this contribution is accentuated in chronic hypoxia either by greater release of adenosine or greater vascular sensitivity to it.
Adenosine↗
The effects of systemic hypoxia on renal function in the anaesthetized rat.
1. In rats anaesthetized with Saffan, renal function was monitored from the left kidney from the 5th minute of spontaneous breathing of 12% O2 for two 20 min periods and during air breathing before, between and after the hypoxic periods. Two groups of animals (I and II) were used, each group comprising two subgroups in which the left kidney was innervated or denervated, respectively; in Group II, renal perfusion pressure (RPP) was maintained during the 2nd hypoxic period by occl97uding the distal aorta. 2. In both subgroups of Group I, both hypoxic periods produced hyperventilation, arterial PO2 falling to approximately 50 mmHg. Concomitantly, mean arterial pressure (MABP) fell by similar extents (approximately 23%, from a baseline level of 140 mmHg during the 2nd hypoxic period). In the innervated subgroup, renal vascular conductance (RVC) increased, but glomerular filtration rate (GFR) fell (by 48 and 6%, respectively, during the 2nd hypoxic period), while urine flow, absolute sodium excretion (UNaV) and fractional sodium excretion (FENa) fell (by 52, 63 and 61%, respectively). Baseline urine flow, UNaV and FENa were higher in the denervated subgroup, but hypoxia produced similar percentage changes from baseline in all variables. 3. In Group II, both subgroups showed similar changes during the 1st hypoxic period as the corresponding subgroups of Group I. However, during the 2nd hypoxic period when the fall in MABP was reduced to approximately 7%, the increase in RVC persisted only in the denervated subgroup; there was no significant change in GFR, urine flow, UNaV or FENa in either subgroup. 4. These results indicate that, in the rat, moderate hypoxia produces antidiuresis and antinatriuresis that are not dependent on the renal nerves, but are dependent on the hypoxia-induced fall in MABP. The fall in renal perfusion pressure (RPP) may directly determine renal function, but reflex influences upon the kidney initiated by, for example, arterial baroreceptor unloading, may play a role. The fall in GFR and increase in RVC, which persisted after denervation or when renal perfusion was controlled, implies a local dilatatory influence acting preferentially on the efferent arterioles.
Animals↗
A study on rats of the effects of chronic hypoxia from birth on respiratory and cardiovascular responses evoked by acute hypoxia.
1. Comparative studies were performed on eighteen rats 54 days old made chronically hypoxic from birth in an hypoxic chamber at 12% O2 (CHB), and in eight weight-matched control rats (NB, 42 days old); both CHB and NB rats were anaesthetized with Saffan. 2. In NB rats, breathing 12 or 8% O2 for 5 min induced a pattern of response comparable to that described in older rats (10-11 weeks old): an initial increase and secondary fall in minute volume (VE), a fall in arterial pressure (ABP), an increase in muscle vascular conductance, while cerebral blood flow (CBF) increased at the 1st minute in six animals and fell by the 5th minute in all animals. The adenosine receptor antagonist 8-phenyl-theophylline (8-PT, 10 mg kg-1) reduced the secondary fall in VE, the fall in ABP and muscle vasodilatation, indicating they were partly mediated by adenosine. 3. In CHB rats breathing 12% O2, VE was higher (277 +/- 12 vs. 204 +/- 18 ml min-1), arterial partial pressures of O2 (45 +/- 2 vs. 88 +/- 3 mmHg), CO2 (32 +/- 1 vs. 44 +/- 1 mmHg) and ABP (105 +/- 5 vs. 131 +/- 5 mmHg) were lower, while muscle vascular conductance was higher (0.08 +/- 0.01 vs. 0.03 +/- 0.01 ml min-1 mmHg-1) than in NB rats breathing air; these differences were reduced, but not abolished, when CHB rats acutely breathed air for 5 min. 4. In CHB rats, the smaller change from 12 to 8% O2 for 5 min evoked a similar pattern of response to that evoked by 8% O2 in NB rats, except that heart rate (HR) and CBF decreased progressively. However, 8-PT increased baseline VE and reduced ABP in 12% O2 and reduced the secondary decrease in VE and HR evoked by 8% O2, but had no effect on the fall in ABP, or change in muscle vascular conductance. 5. We propose that in CHB rats (i) there is accentuation of the components of the response to acute hypoxia (the fall in ABP, HR and CBF) that form a positive feedback loop which promotes central ventilatory depression and (ii) that adenosine exerts a tonic inhibitory influence on VE and vasodilator influence in muscle and mediates the secondary fall in VE, but not the muscle vasodilation induced by acute hypoxia.
Adenosine↗