James A. Orr

Cardiopulmonary function in exercising bar-headed geese during normoxia and hypoxia

To investigate possible physiologic mechanisms that allow the bar-headed goose to perform strenuous physical activity when flying at high altitude (e.g., above 9,000 m), we measured cardiopulmonary variables during running exercise (treadmill; 0.6 m.sec-1; 2 degrees incline) while the bird breathed either normoxic (21% O2) or hypoxic (7% O2) gases via a face mask. 1. During normoxic exercise, O2 uptake rate doubled and both ventilation and cardiac output increased. Blood gases and pH in arterial, mixed venous and blood from the leg, however, remained virtually unaltered. 2. Hypoxia at rest stimulated ventilation to rise but not cardiac output. The birds reached a steady state with virtually unaltered O2 uptake. 3. Exercise during hypoxia further stimulated ventilation, resulting in elevated arterial PO2 and O2 content compared to hypoxia at rest. However, O2 uptake increased only slightly, and cardiac output did not rise over the resting hypoxic value. The hyperventilation resulted in respiratory alkalosis and increased CO2 output, with R values being as high as 2.0. 4. It is concluded that neither ventilation nor pulmonary gas transfer were the limiting step in supplying O2 to the working muscles during hypoxic exercise in our experiments. It is more likely that muscle blood flow or diffusion from muscle capillaries to mitochondria, or both, determined the aerobic capacity under these conditions.

Stimulation of group III and IV afferent nerves from the hindlimb by thromboxane A2

These experiments were designed to test the hypothesis that the TxA2 mimetic, U46,619, would stimulate group III and IV afferent nerve endings from the hindlimb of the anesthetized cat. Nerve impulses were recorded from the dorsal rootlets of the L7-S1 segments of the spinal cord, and afferent units identified by measurement of conduction velocities, mechanical probing of hindlimb muscles, and local injection of chemical stimulants (capsaicin and bradykinin). Five of the 15 group III fibers were stimulated by U46,619 (2-10 micrograms injected into the abdominal aorta; mean baseline impulse frequency increasing from 7.3 (+/- 3.2) impulses/s to 16.0 (+/- 3.1)), while 7 of the 12 group IV fibers responded to U46,619 (impulse frequency increasing from 4.3 (+/- 3.2) to 8.8 (+/- 3.6)). The average latency for the response (20-30 s) did not differ between the two groups of afferent fibers. We conclude that group III and IV afferent fibers originating from the skeletal muscle of the hindlimb are stimulated by TxA2 and that the release of TxA2 in skeletal muscle could evoke cardiorespiratory reflexes known to be activated by stimulation of these afferent nerves.

Inhibition of Thromboxane A2-Induced Arrhythmias and Intracellular Calcium Changes in Cardiac Myocytes by Blockade of the Inositol Trisphosphate Pathway

We have recently reported that left atrial injections of the thromboxane A2 (TXA2) mimetic, (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA2 may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA2R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA2R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA2R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP3) formation, gentamicin, or with an inhibitor of IP3 receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA2 can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP3 pathway. In summary, this study provides novel evidence for direct TXA2-induced cardiac arrhythmias and provides a rationale for IP3 as a potential target for the treatment of TXA2-mediated arrhythmias.

Flow cytometry and laser scanning cytometry, a comparison of techniques

ObjectiveFlow and laser scanning cytometry are used extensively in research and clinical settings. These techniques provide clinicians and scientists information about cell functioning in a variety of health and disease states. An in-depth knowledge and understanding of cytometry techniques can enhance interpretation of current research findings. Our goal with this review is to reacquaint clinicians and scientists with information concerning differences between flow and laser scanning cytometry by comparing their capabilities and applications.MethodsA Pubmed abstract search was conducted for articles on research, reviews and current texts relating to origins and use of flow and laser scanning cytometry. Attention was given to studies describing application of these techniques in the clinical setting.ResultsBoth techniques exploit interactions between the physical properties of light. Data are immediately and automatically acquired; they are distinctly different. Flow cytometry provides valuable rapid information about a wide variety of cellular or particle characteristics. This technique does not provide the scanned high resolution image analysis needed for investigators to localize areas of interest within the cell for quantification. Flow cytometry requires that the sample contain a large amount disaggregated, single, suspended cells. Laser scanning cytometry is slide-based and does not require as large of a sample. The tissue sample is affixed to a slide allowing repeated sample analyses. These cytometry techniques are used in the clinical setting to understand pathophysiological derangements associated with many diseases; cardiovascular disease, diabetes, acute lung injury, hemorrhagic shock, surgery, cancer and Alzheimer’s disease.ConclusionsUnderstanding the dif- ferences between FCM and LSCM can assist investigators in planning and design of their research or clinical testing. Researchers and clinicians optimize these technique capa- bilities with the cellular characteristics they wish to measure delineating molecular and cellular events occurring in health and disease. Discovery of mechanisms in cells using FCM and LSCM provide evidence needed to guide future treatment and interventions.

Cardiopulmonary responses to HCl infusion are mediated by thromboxane A2 but not by serotonin.

Intravenous infusion of HCl has been shown to elicit the release of thromboxane A2 (TxA2) which alters blood pressure and breathing independent of reductions in circulating blood pH. The present experiments were designed to determine if the release of serotonin (5-HT) in the anesthetized cat contributed to cardiorespiratory responses during acid infusion and, furthermore to define the source of TxA2, viz. blood or other tissues. To infuse HCl into the bloodstream without reducing circulating blood pH (= neutral acid-base infusion), an extracorporeal arteriovenous shunt (20 ml/min) between the femoral artery and femoral vein was installed. Into this loop, acid (0.25 M HCl), and approximately 10 cm downstream, base (0.25 M NaOH) could be infused whereby blood pH could be locally reduced in the blood within the loop. This procedure was performed in three groups of cats: one group which received no drugs, a second group that was pretreated with indomethacin (2.5 mg/kg) and a third group that received the 5-HT2 receptor antagonist, ketanserin (0.75 mg/kg), prior to the infusion. During neutral acid-base infusion in the nontreated animals, right ventricular blood pressure (PRV) increased and systemic arterial blood pressure (Pa) decreased. Respiratory frequency was increased, but total ventilation was not elevated because of a concomitant fall in tidal volume (VT). The response was transient and could not be evoked with repetitive infusions of HCl and NaOH. These responses were significantly attenuated in the indomethacin-treated animals, but persisted in the cats pretreated with ketanserin. In addition, TxB2, the stable degradation metabolite of TxA2, was elevated during the acid/base infusion, but there were no measurable changes in plasma 5-HT concentration. The source of TxA2 was likely to be the blood since TxB2 was increased in plasma when acid and base were added to blood in vitro. We conclude from these experiments that transient cardiorespiratory responses to HCl infusion are mediated by the release of TxA2 from the blood and do not involve serotonin.

Detection of thromboxane A2 receptor mRNA in rabbit nodose ganglion neurons

Thromboxane A(2) (TXA(2)) is an arachidonic acid metabolite that is released during tissue trauma and elicits platelet aggregation and vascular smooth muscle contraction. Previous research has shown that TXA(2) stimulates pulmonary and cardiac vagal afferent neurons. Therefore, we hypothesized that the presence of the TXA(2) receptor (TP) in vagal neurons would allow for stimulation or modulation of these neurons by TXA(2). To test this hypothesis, single cell RT-PCR was employed using neurons obtained from primary cell cultures of nodose ganglia excised from adult rabbits. Since the sequence for the rabbit TP gene was unknown, a portion of the rabbit TP cDNA was first amplified, cloned, and sequenced. Primer sets for TP were then designed based on this sequence and used in conjunction with a neuronal marker, medium weight neurofilament (NFM), in multiplex RT-PCR reactions. Ninety-three cells were isolated from culture and RT-PCR was carried out on individual cells. Using an aliquot from the initial RT-PCR reaction, a second round of PCR was then employed in which the NFM and TP primer sets were split up into separate reactions. Twenty-three of the 82 cells that were positive for NFM were also positive for TP. Therefore, we conclude that the presence of TP mRNA in a subset of cultured nodose ganglion neurons allows for the possibility that TXA(2) may directly stimulate or modulate vagal afferent neurons.

The Role of Protein Kinase C in Thromboxane A2-Induced Pulmonary Artery Vasoconstriction

In order to determine if protein kinase C (PKC) plays a significant role in the stimulant action of thromboxane A2 (TxA2) on pulmonary vascular smooth muscle, TxA(2)-induced contractile responses were measured following inhibition of PKC. Rabbits were sacrificed and segments of the main trunk of the pulmonary artery were removed and placed within a temperature-controlled (37 degrees C) organ bath. Contractile responses that were evoked by a TxA2 mimetic (U46,619, 0.5 microM) decreased by 27 and 35% following treatment with the PKC inhibitors, calphostin C (2 microM) and staurosporine (200 nM), respectively. These results account for the effect of the vehicle, DMSO, which was also found to have a concentration-dependent inhibitory effect on the U46,619-induced contractions. The effects of DMSO alone was subsequently subtracted from the previously measured responses to PKC inhibitors that were dissolved in DMSO to obtain effects attributable to the PKC inhibitor alone. It can therefore be concluded that inhibition of PKC results in partial attenuation of U46,619-induced responses supporting the hypothesis that activation of PKC plays a partial role in TxA2-induced contraction of pulmonary arterial smooth muscle.

TxA2-induced pulmonary artery contraction requires extracellular calcium.

In order to examine the role of extracellular calcium in the pulmonary arterial vasoconstriction that is elicited by thromboxane A2 (TxA2), rabbits were sacrificed and the main trunk of the pulmonary artery removed. Contractile responses of the isolated vessel to the TxA2 mimetic, U46 619, were measured in a temperature controlled (37 degrees C) organ bath. Compared with control responses, U46 619 microM) contractions were nearly eliminated when 1 mM EGTA was added to the buffer. In the presence of normal extracellular calcium concentrations, antagonists of voltage sensitive calcium channels (e.g. verapamil and nifedipine) attenuated the U46 619-induced contractions. These voltage sensitive calcium channel blockers were more effective in eliminating contractile responses to high KCl concentrations (6) or 120 mM KCl). The inability of these calcium channel antagonists to completely eliminate U46 619 responses was confirmed in the anesthetized rabbit where both nifedipine and verapamil failed to block the increase in pulmonary arterial blood pressure resulting from intravenous U46 619 infusion. These results indicate that extracellular calcium is essential for U46 619-induced pulmonary vascular contraction, and that mechanisms in addition to voltage operated calcium channels participate in the movement of extracellular calcium through the plasma membrane.

Thromboxane A2 mimetic, U46,619, and slowly adapting stretch receptor activity in the rabbit

The effect of infusing the thromboxane A2 mimetic U46,619 on afferent activity from slowly adapting airway stretch receptors (SARs) in the anesthetized rabbit was examined in these experiments. SAR vagal afferent fibers (n = 29) were identified by their slow adaptation to a sustained (10-15 s duration) lung inflation in the closed-chest, mechanically ventilated animal (n = 16). Intravenous infusion of U46,619 increased the discharge frequency of the SAR, measured at the end of inspiration, in a dose-dependent manner: by 6.6% and 8.0% at doses of 0.1 and 0.5 microgram of U46,619/kg, respectively. This increase in SAR activity was correlated with increases in end-inspiratory tracheal airway pressure (6.4% at 0.1 micrograms/kg and 9.1% at 0.5 micrograms/kg). Higher doses of U46,619 could not be administered due to decreases in systemic arterial blood pressure. The increase in SAR activity and tracheal airway pressure was qualitatively comparable to the response to histamine (25 micrograms/kg), a known bronchoconstrictor. We conclude that intravenous infusion of U46,619 in the anesthetized rabbit at doses that elicit significant hemodynamic effects causes modest bronchoconstriction and comparable increases (less than 10%) in SAR afferent nerve activity. From these data, it appears that U46,619 has no direct effect on SARs, but rather increases SAR activity due to bronchoconstriction.

Cerebrovascular response to acute decreases in arterial PO2

The purpose of these studies was to examine the time course of the cerebrovascular response to acute hypoxia in unanesthetized ponies. An electromagnetic flow transducer chronically placed on the internal carotid artery of the pony allowed continuous recording of internal carotid artery blood flow (ICBF) which has been shown to be representative of cerebral blood flow (CBF). The ponies were subjected to three levels of acute isocapnic hypoxia (PaO2 = 62, 44, and 39 mm Hg for hypoxia level I, II, and III, respectively), and the temporal and steady-state cerebrovascular response was examined. ICBF increased significantly at all three hypoxia levels (8, 25, and 40% at hypoxia I, II, and III, respectively). This increase was rapid in the two most severe levels of hypoxia, beginning within 45 s, and was complete within 90 s. The increase lagged behind the reduction in PaO2 by 24–28 s. During the very mild level of hypoxia (I), no such rapid increase in flow was observed; rather, the increase occurred only after 5 min of hypoxia. Microsphere (15 μm diameter) measurements from six ponies during the most severe level of hypoxia (III) demonstrated that CBF increased 38%. Noncerebral tissues known to be vascularly connected to the circle of Willis, and thus capable of receiving blood flow via the internal carotid artery, either did not change or increased so slightly during hypoxia that their effect on ICBF was minimal. These data imply that mediators of the cerebrovascular response to hypoxia must be capable of sensing hypoxia and affecting cerebrovascular smooth muscle within seconds. The slower time course in mild hypoxia suggests other mechanisms may be involved when PaO2 is greater than 50 mm Hg.