Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation.

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted Msh2 alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from Msh2(+/-) and Msh2(-/-) mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from Msh2(+/+) mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.

Regional circulatory responses to intestinal work in developing swine.

Circulatory effects of intraduodenal feeding with 2 and 5% glucose were studied in 29 fasted swine (1 day to 1 mo old) anesthetized with pentobarbital. Recordings included aortic and intestinal venous pressures and intestinal, renal, and femoral blood flows. Calculations included vascular resistances, arterial and intestinal venous O2 contents, and intestinal O2 consumption. Observations were made before and at 15 and 30 min after a feeding and at end of experiments. Blood flow autoregulation was evaluated before and after feedings. Glucose induced increases in intestinal O2 consumption and blood flow at all ages, but intestinal blood flow autoregulation was enhanced only in 2 wk olds. Blood flow was redistributed to the working gut from the hindlimb, but not the kidney, at all ages. Renal blood flow autoregulation was sustained in 2-wk-old and 1-mo-old animals and became significant in 1 wk olds during intestinal hyperemia. We concluded that basic mechanisms governing blood flow redistribution from hindlimb to working gut are available at birth in swine and that maintenance of renal blood flow depends only partly on autoregulatory capability.

Regional circulatory responses to hindlimb work in developing swine.

Circulatory effects of hindlimb work were studied in 29 swine (aged 1 day to 1 month) anesthetized with pentobarbital. Femoral, renal and intestinal blood flow, resistance and autoregulatory capability were determined at different levels of hindlimb oxygen consumption before and during distal sciatic nerve stimulation. Increases in oxygen consumption were accompanied by hyperemia at all ages, but by increased oxygen extraction only in 2-week and 1-month-old animals, without evidence of autoregulation. Blood flow was consistently redistributed from the kidney and small intestine only in neonates. Older animals generally sustained autoregulation of renal and intestinal blood flows throughout experiments. We concluded that hindlimb work evokes age-related adjustments in regional blood flow that reflect the balance of neural and autoregulatory control.

Histamine or adenosine blockade alters intestinal blood flow autoregulation in swine.

The possible role of histamine or adenosine in intestinal blood flow autoregulation in 1-mo-old swine was examined by obtaining pressure-flow relationships before and during intestinal histamine H1- or adenosine-receptor blockade in two groups of fasting animals under anesthesia with pentobarbital sodium (30 mg/kg). Changes in abdominal and thoracic aortic pressures and in superior mesenteric and left renal arterial flows were recorded during controlled aortic compression above the celiac artery. After control intestinal and renal pressure-flow relationships were obtained, a test dose of agonist (0.1 microgram histamine or 0.2 microgram adenosine/kg body wt) was given into the superior mesenteric artery. Then an intra-arterial infusion of blocking agent was started (0.1 mg.kg-1.min-1 chlorpheniramine or 10 mumol/min theophylline). Degree of blockade was assessed with doses of agonist given before and after a second set of intestinal and renal pressure-flow relationships was obtained. Complete blockade of intestinal vascular histamine H1-receptors with chlorpheniramine abolished, and incomplete blockade of adenosine-receptors with theophylline attenuated, intestinal blood flow autoregulation. Renal blood flow autoregulation remained at its control level. These results indicate that both histamine and adenosine are among the physiological vasodilators contributing to intestinal blood flow autoregulation when arterial pressure is decreased in young swine.

Vasoactive compound effects on autoregulating versus nonautoregulating intestinal and renal circulations in young swine.

Vascular resistance changes to single intra-arterial injections of norepinephrine, histamine and adenosine were examined in 12 kidney and 10 jejunum preparations perfused in situ in fasting swine anesthetized with pentobarbital. Threshold doses were higher and other response magnitudes were smaller in the nonautoregulating renal circulation of 1-week-olds and jejunal circulation of 2-week-olds than in autoregulating circulations of 1-month-olds. These results suggest a correlation between maturation of autoregulatory capability and of vasodilator histamine and adenosine receptors.

Autoregulatory escape from vasoconstriction of intestinal circulation in developing swine.

The capability of the developing intestinal circulation to maintain a vasoconstrictor response during postganglionic adrenergic nerve stimulation or norepinephrine infusion was examined in 34 swine aged 6 h to 2 mo anesthetized with pentobarbital sodium. Aortic and portal venous pressures, electrocardiogram (ECG), and blood flow (F) through the superior mesenteric artery were recorded, and intestinal vascular resistance (MVR) was calculated as mean pressure difference per mean F. Baroreceptor reflex inhibition by bilateral occlusion of the carotid arteries increased MVR, section of the splanchnic nerve and postganglionic fibers decreased MVR, and short-latency F decreases were obtained during mesenteric nerve stimulation (MNS). Latencies for the decreases in F shortened with age and with increasing MNS frequency (5-17 Hz) at any age. Prolonging MNS for 60 s at 10 or 12 Hz led to sustained high MVR in 6-h to 7-day-old animals; however, MVR decreased toward control before the end of the 60-s MNS period in animals 1 to 2 mo old. Intra-arterial infusion of norepinephrine (0.5 microgram X kg-1 X min-1) decreased F and increased MVR in all animals; but by 5 min of infusion, F was returning toward control level in all but the youngest. This demonstration that the least mature intestinal circulation was least capable of autoregulatory escape from vasoconstriction provides further evidence of its functional immaturity.

Intestinal and femoral blood flow autoregulation in developing swine.

Pressure-flow relationships (P/F) in the small intestine or hindlimb circulation were used to examine autoregulation in 104 swine, aged 1 day to 2 months and anesthetized with pentobarbital. The P/F were obtained while perfusion pressure was decreased by compression of the abdominal aorta for 2 min at each pressure. Readjustments in vascular resistance stabilized within 2 min. Intestinal flow decreased with each decrease in pressure in swine 2 weeks old or younger, but was well maintained by 1 month of age. Femoral flow decreased with each decrease in pressure at all ages studied. Control experiments indicated that the observed vascular responses were independent of angiotension-II activity and were not reflex in origin.

Maturation of circulatory system in three mammalian models of human development.

The review surveys the literature on maturation of vasoconstrictor and vasodilator functions in cerebral, renal and intestinal circulations of three non-primate models of human development. An ovine model has been refined for use at both fetal and neonatal stages of development. Important variables controlling regional circulations in the lamb fetus at term include arterial O2 content and pCO2 (brain), angiotensin-II (kidney) and norepinephrine (small intestine). Blood flow autoregulation to decreasing perfusion pressure has been inferred for the renal circulation of the neonate. A canine model has been employed in the postnatal period, usually later than the first week after birth. Important variables controlling regional circulations in the young puppy include arterial pO2 and pCO2 (brain) and epinephrine and angiotensin-II (kidney). Blood flow autoregulation to decreasing pressure has been demonstrated in the cerebral circulation at birth and in the renal circulation at one week thereafter. The intestinal circulation has not been studied with respect to blood flow control. A porcine model has been examined from birth through at least two months of postnatal life. Important variables controlling regional circulations in swine at birth include adrenergic nerve stimulation, arterial pCO2 (brain), angiotensin-II (kidney) and norepinephrine (kidney and small intestine). Blood flow autoregulation to decreasing perfusion pressure has been demonstrated in the brain by the fourth day, in the kidney by the end of the second week and in the small intestine by the end of the first month after birth. The advantage of each model for further investigation of functional maturation of regional circulatory control is summarized.

Mechanical properties of developing swine myocardium.

Mechanical responses of myocardium from 16 piglets were studied from 18 hr to 12 days after birth. Tension, time and velocity parameters of contraction and relaxation were determined for every contraction cycle. Increasing the frequency of stimulation in step-changes induced negative inotropy in some muscles regardless of age. Doubling extracellular calcium ion concentration induced a positive force-frequency response in all muscles. Epinephrine increased tension and velocities without affecting contraction time. The ultrastructure was immature even on the 12th postnatal day. We concluded that in newborn piglet hearts, the mechanisms for calcium delivery are not fully developed. Thus, the heart undergoes a transient phase during which at least a principal portion of calcium for the myofibers is supplied by the extracellular fluid. While receptors for catecholamines are present, the time course for their response is immature.

Circulatory effects of splanchnic nerve stimulation in developing swine.

The relative maturity of immediate intestinal circulatory responses to efferent splanchnic nerve stimulation for 20 s was tested in 34 piglets (1 day-1 mo old) under pentobarbital anesthesia (15-30 mg/kg). Aortic pressure, heart rate, and superior mesenteric arterial flow (measured by electromagnetic flow transducers) were recorded simultaneously. Intestinal vascular resistance was calculated as mean aortic pressure/mean arterial flow. Resistance increased in most animals during experimental inhibition of the carotid sinus baroreceptor reflex. Transection of the splanchnic nerve decreased mesenteric resistance in all animals. Nerve stimulation at 2 Hz was not effective in all of the youngest animals; at 3 Hz, mesenteric resistance increased in all animals. The latency of this response was shorter at each increase in stimulation frequency. The aortic pressure rise associated with splanchnic nerve stimulation was greater with each increase in frequency. This rise was attenuated or absent after adrenalectomy and during postganglionic fiber stimulation. The vasoconstrictor effect of splanchnic nerve stimulation was attenuated after alpha-adrenergic receptor blockade with phentolamine in a dose (0.25-0.75 mg/kg) that blocked vasoconstrictor effects of norepinephrine (0.5 micrograms/kg). We conclude that alpha-adrenergic mechanisms are functional, although not mature, and that sympathetic vasoconstrictor tone is present in the intestinal circulation of swine at birth.

Cardiovascular and renal effects of isoproterenol infusions in young swine.

The cardiovascular and renal effects of intravenous (i.v.) and intra-arterial (i.a.) infusions of isoproterenol (ISP, 0.1-0.2 micrograms/kg/min) were evaluated in 17 two-week-old swine anesthetized with pentobarbital. The glomerular filtration rate (GFR) of each kidney and blood flow and vascular resistance (RVR) of the left kidney were determined in all animals. In the 8 animals given ISP i.v., right ventricular pressure and dP/dtmax were also determined via a thoracotomy. In 9 animals, ISP was given i.a. after stabilization of constant-flow perfusion of the left kidney in situ. During i.v. infusion of ISP, the positive inotropic and chronotropic effects and the decrease in arterial pressure were maintained; renal blood flow and GFR increased and RVR decreased. During i.a. infusion of ISP in the constant-flow perfused kidney, similar changes in RVR and GFR were observed despite the higher effective concentrations of drug reaching the kidney. We conclude that, at this stage of postnatal renal development, the infusion of cardiotonic doses of ISP lowers RVR and produces a small increase in GFR.

Renal blood flow autoregulation in developing swine.

Pressure-flow relationships (P/F) in the renal circulation were determined in 62 swine, aged 1 day-2 mo, anesthetized with pentobarbital sodium. Aortic and inferior vena caval pressures and renal and femoral arterial flows were recorded. Blood gas composition and pH and body temperature were monitored. The P/F was first determined while perfusion pressure was decreased for 2 min at each pressure by suprarenal aortic occlusion. The left renal artery in 38 of these animals was then cannulated for in situ perfusion of the kidney with blood withdrawn from a carotid artery by a Masterflex pump. The P/F was subsequently determined by changing pump flow for 2 min at each flow while recording perfusion pressure. Records were analyzed for transient and steady-state effects. Readjustments in renal vascular resistance (RVR) were apparent within 5 s after changing pressure or flow. The RVR stabilized at a new level within 2 min. Graphs of steady-state data delineated an autoregulatory range in the P/F for animals as young as 2 wk of age. We conclude that renal blood flow autoregulation in this mammal is negligible at birth and develops progressively during the first postnatal month.

Maturation of circulatory responses to adrenergic stimuli.

The postnatal maturation of renal and femoral circulatory responses to catecholamines and to stimulation of their efferent sympathetic nerve supply has been examined in developing swine. Catecholamine dose-response experiments were carried out in intact animals under pentobarbital anesthesia. Effects of denervation and nerve stimulation were studied in intact animals and effects of neurotransmitter infusions were studied in preparations for in situ perfusion of kidney or hind limb. The renal circulation was found to be relatively sensitive to norepinephrine and under tonic neural vasoconstrictor influence at birth, and the femoral circulation was not. The femoral circulation was found to be relatively sensitive to isoproterenol at birth, whereas the renal circulation exhibited beta-adrenergic vasodilation by the end of the first postnatal week. The basic pressure-flow relationship in either circulation at any age was not altered by infusions of neurotransmitters into the arterial circuit after denervation of the perfused kidney or hind limb. Adrenergic innervation continued to mature rapidly in both circulations during the first postnatal week, as evidenced by the decreasing threshold and increasing magnitude of vasoconstrictor responses to electrical stimulation of the renal or lumbar nerves. A cholinergic component of the response to lumbar nerve stimulation became functional at 1 month after birth.

Cardiovascular effects of dopamine in developing swine.

Cardiac function and peripheral blood flows were measured before and during single intravenous injections of dopamine (2-25 micrograms/kg) in developing and mature swine anesthetized with pentobarbital. A positive inotropic effect was observed in even the youngest swine. Renal vasoconstriction was observed even after low doses of dopamine in animals younger than 1 month of age, and femoral vasoconstriction in animals younger than 2 weeks of age, unless alpha-adrenergic receptors were blocked by phentolamine. We concluded that the vasodilator responses to dopamine are slower to develop in the renal than in the femoral circulation.

Femoral circulatory responses to lumbar nerve stimulation in developing swine.

The maturation of femoral circulatory responses to efferent lumbar nerve stimulation was tested in 51 developing swine (1 day-3 mo old) under pentobarbital sodium anesthesia (10-30 mg/kg). Aortic pressure, heart rate, and femoral and carotid arterial flows (measured by electromagnetic flow transducers) were recorded simultaneously. Femoral vascular resistance was calculated as mean aortic pressure/mean flow. Transection of the lumbar nerve fibers below the last ganglion in the sympathetic chain did not after femoral resistance in day-old animals but decreased femoral resistance in swine 1 wk of age and older. Efferent lumbar nerve stimulation at various combinations of frequencies and intensities revealed an atropine-blockable vasodilator component in the femoral circulatory response in swine 1 mo of age and older. After alpha-adrenergic receptor blockade with phentolamine (0.25 or 0.5 mg/kg), femoral vasodilation occurred during low-frequency and -intensity stimulation of the lumbar nerve only in animals 1 mo of age and older. Acetylcholine (2 micrograms ia) caused a decrease in femoral resistance at all ages. Vasoconstrictor effects of high-frequency stimulation (5-10 Hz) were present at all ages and were age dependent. The results of these experiments suggest that the femoral circulation in swine at birth in innervated by functionally active vasoconstrictor fibers, which do not provide a tonic influence on femoral resistance until late in the first postnatal week. Furthermore, although femoral vascular cholinergic receptors are demonstrable at birth, there appears to be a delay in the maturation of functionally active vasodilator fibers.

Changes in cardiovascular and renal function during catecholamine infusions in developing swine.

Renal and cardiac effects of norepinephrine and dopamine were evaluated in swine aged 1 wk, 2 wk, and 6 mo. The swine were anesthetized with pentobarbital (20-30 mg/kg). Aortic pressure, right ventricular pressure and its first derivative, and heart rate were recorded, together with carotid and renal (RBF) arterial flows. Glomerular filtration rate (GFR) was determined by [14C]inulin clearance. After a control period, norepinephrine or dopamine was infused intravenously for 10-20 min before and then during another clearance period. After a second control period, the second catecholamine was infused. GFR increased in piglets given either catecholamine. Norepinephrine at equipressor doses (2.0 micrograms.kg-1.min-1 in piglets and 1.0 micrograms.kg-1.min-1 in mature swine) decreased RBF and increased renal resistance. Dopamine at equi-inotropic doses (10 micrograms.kg-1 min-1 in piglets and 20 micrograms.kg-1.min-1 in mature swine) increased RBF and decreased renal resistance only in mature swine. Infusions of dopamine at a low dose (5 micrograms.kg-1.min-1) also failed to increase RBF or decrease renal resistance in piglets. The results suggest that maturation of the mechanism of renal vasodilation by dopamine occurs later than that for vasoconstriction by norepinephrine.

Renal circulatory effects of adrenergic stimuli in anesthetized piglets and mature swine.

The relative maturity of renal circulatory responses to efferent renal nerve stimulation, and to exogenous norepinephrine and isoproterenol, was tested in 62 piglets (1--16 days old) under pentobarbital anesthesia (10--25 mg/kg). Aortic pressure, heart rate, and renal and femoral arterial flows (measured by electromagnetic flow transducers) were recorded simultaneously. Renal vascular resistance was calculated as mean aortic pressure/mean flow. Transection of the renal nerve resulted in decreased renal resistance in all animals. Efferent renal nerve stimulation at increasing frequencies (2--12.5 Hz, at 1.2 ms pulse duration and 1.0 mA current) showed age-dependent differences in the threshold and also in the magnitude of increase in renal resistance. Norepinephrine (0.05--1.0 microgram/kg) caused age-dependent increases in renal resistance. Restoration of renal flow toward control level occurred during the peak pressor effect of norepinephrine only in older piglets. Isoproterenol (0.05--1.0 microgram/kg) did not alter renal resistance consistently in piglets younger than 1 wk. Phentolamine (0.25 mg/kg) attenuated or blocked resistance increases to 0.5 microgram norepinephrine/kg or to renal nerve stimulation at 12.5 Hz in all animals. Propranolol (0.1 mg/kg) attenuated or blocked resistance decreases to 0.1 microgram isoproterenol/kg, which occurred only in older piglets. These results indicate the presence of an active alpha-adrenergic vasoconstrictor mechanism and absence of the beta-adrenergic vasodilator mechanism in the renal circulation of swine at birth.

Age-related cardiovascular effects of catecholamines in anesthetized piglets.

We studied cardiac and peripheral circulatory effects of graded doses of catecholamines (0.05-1.0 microgram/kg) in piglets aged less than or equal to 1 day, 2--4 days, 1 wweek, 2 weeks, and 2.5-3 months, under anesthesia with pentobarbital. We evaluated cardiovascular function from simultaneous recordings of aortic pressure, ventricular pressure and its first derivative, heart rate, and phasic carotid and femoral blood flows. We calculated vascular resistance as the ratio of mean aortic pressure to mean flow. The age of onset of a given cardiovascular response was determined, and magnitudes of each type of response were compared among the age groups. Norepinephrine elevated the blood pressure at all doses in piglets of all ages, elicited reflex bradycardia only in older piglets, and increased carotid resistance. Epinephrine elevated the blood pressure at all doses in piglets less than 1 week old, but low doses lowered the blood pressure in piglets older than 1 week of age; resistance changes in the femoral and carotid circulations were variable except in the 2.5-3 month age group. Isoproterenol increased cardiac contractility at all doses in piglets of all ages and increased heart rate at low doses in piglets older than 2 days of age; however, blood pressure and femoral resistance decreases were age and dose dependent. There were age-related differences in the catecholamine dose required to elicit a given cardiac or peripheral circulatory effect and age-related differences in the direction and magnitude of such effects. These results provide evidence for differing rates of postnatal maturation of cardiovascular alpha- and beta- adrenergic mechanisms in swine.

Cardiovascular function in anesthetized miniature swine.

Miniature swine anesthetized with pentobarbital were studied with respect to their cardiovascular function under control conditions and in response to catecholamines, baroreceptor inhibition, bilateral vagotomy and vagal nerve stimulation. Measurements included aortic pressure, heart rate, intraventricular pressure and its maximum rate of rise during contraction, carotid blood flow and resistance, femoral blood flow and resistance, and renal blood flow and resistance. The cardiovascular actions of norepinephrine, epiniphrine and isoproterenol were similar to those in other mammals, and the adrenergic receptor mechanisms also were susceptible to blockade with phentolamine or propranolol. Inhibition of the carotid baroreceptors was accompanied by elevation of aortic pressure, reflex bradycardia and increased femoral and renal resistances. Bileteral vagotomy was followed by hypertension, tachycardia and increased renal resistance. Changes in femoral resistance to these procedures differed between the two strains of miniature swine studied. Stimulation of the peripheral end of either vagus nerve was accompanied by bradycardia without hypotension.