Capillary and arteriolar responses to local vasodilators are impaired in a rat model of sepsis

Although sepsis isknown to affect vascular function, little is known about changes at thecapillary level. We hypothesized that sepsis attenuates the"upstream" arteriolar response to vasoactive agents appliedlocally to capillaries. Sepsis in rats was induced by cecal ligationand perforation. After 24 h, extensor digitorum longus muscle wasprepared for intravital microscopy. Phenylephrine (PE, 10 mM) andacetylcholine (ACh, 10 mM) were applied iontophoretically on terminalarterioles and on their downstream daughter capillaries (300 µm fromarteriole). There was no significant difference between control andseptic rats in baseline arteriolar diameters [8.0 ± 0.6 vs.9.8 ± 0.8 (SE) µm] or baseline red blood cellvelocity (V_RBC)in perfused daughter capillaries (255 ± 10 vs. 264 ± 13 µm/s). Application of PE onto arterioles resulted in comparable constrictions (i.e., 22% diameter change) andV_RBC reductions (100%) in control and septic rats. In contrast, arteriolardiameter and V_RBCincreases after application of ACh were attenuated in sepsis (diameter:from 41 to 14%;V_RBC: from 67 to24%). Application of PE onto the capillary reducedV_RBC to the samelevel (100%) in both groups, whereas application of AChincreased V_RBCless in septic than in control rats (20 vs. 73%). On the basis ofarteriolar-capillary pair stimulations, sepsis affectedV_RBC responses toACh more in the capillary than in the arteriole. When the adenosineanalog 5'-N-ethylcarboxamidoadenosine(0.1 mM) was used instead of ACh, similar effects of sepsis were seen.To test for a possible involvement of inducible NO synthase (iNOS) insepsis-induced attenuated ACh responses, arterioles and capillaries inseptic animals were locally pretreated with the iNOS blockeraminoguanidine (10 mM). In both microvessels, aminoguanidine restoredthe ACh response to the control level. We conclude that impairedcapillary V_RBCand arteriolar diameter responses to vasodilators applied tocapillaries in septic rat skeletal muscle were due to dysfunction atarteriolar and capillary levels. The study underscores the significantrole iNOS/NO may play in sepsis-induced alteration of vascularreactivity in vivo.

     

Effect of hypoxia on pulmonary blood flow-segmental vascular resistance relationship in perfused cat lungs

To investigate the effect of alveolar hypoxia onthe pulmonary blood flow-segmental vascular resistance relationship, wedetermined the longitudinal distribution of vascular resistance whileincreasing blood flow during hyperoxia or hypoxia in perfused catlungs. We measured microvascular pressures by the micropipetteservo-null method, partitioned the pulmonary vessels into threesegments [i.e., arterial (from main pulmonary artery to 30- to50-µm arterioles), venous (from 30- to 50-µm venules to leftatrium), and microvascular (between arterioles and venules)segments] and calculated segmental vascular resistance. Duringhyperoxia, total resistance decreased with increased blood flow becauseof a reduction of microvascular resistance. In contrast, duringhypoxia, not only microvascular resistance but also arterial resistancedecreased with increase of blood flow while venous resistance remainedunchanged. The reduction of arterial resistance was presumably causedby arterial distension induced by an elevated arterial pressure duringhypoxia. We conclude that, during hypoxia, both microvessels andarteries >50 µm in diameter play a role in preventing furtherincreases in total pulmonary vascular resistance with increased bloodflow.

     

Contractile function of single muscle fibers after hindlimb unweighting in aged rats

Thompson, L. V., and J. A. Shoeman. Contractilefunction of single muscle fibers after hindlimb unweighting in aged rats. J. Appl. Physiol. 84(1):229-235, 1998.This investigation determined how muscle atrophyproduced by hindlimb unweighting (HU) alters the contractile functionof single muscle fibers from older animals (30 mo). After 1 wk of HU,small bundles of fibers were isolated from the soleus muscles and thedeep region of the lateral head of the gastrocnemius muscles. Singleglycerinated fibers were suspended between a motor lever and forcetransducer, functional properties were studied, and the myosin heavychain (MHC) composition was determined electrophoretically. After HU, the diameter of type I MHC fibers of the soleus declined (88 ± 2 vs. 80 ± 4 µm) and reductions were observed in peak active force (47 ± 3 vs. 28 ± 3 mg) and peak specific tension(P_o; 80 ± 5 vs. 56 ± 5 kN/m²). The maximal unloadedshortening velocity increased. The type I MHC fibers from thegastrocnemius showed reductions in diameter (14%), peak active force(41%), and P_o (24%), whereas thetype IIa MHC fibers showed reductions in peak active force andP_o. Thus 1 wk ofinactivity has a significant effect on the force-generating capacity ofsingle skeletal muscle fibers from older animals in a fibertype-specific manner (type I MHC > type IIa MHC > type I-IIa MHC).The decline in the functional properties of single skeletal musclefibers in the older animals appears to be more pronounced than what hasbeen reported in younger animal populations.

     

Guinea pig pulmonary hypertension caused by cigarette smoke cannot be explained by capillary bed destruction

Yamato, H., J. P. Sun, A. Churg, and J. L. Wright.Guinea pig pulmonary hypertension caused by cigarette smoke cannot be explained by capillary bed destruction. J. Appl.Physiol. 82(5): 1644-1653, 1997.Chronic exposureto cigarette smoke is known to produce pulmonary hypertension in humansand in animal models, but the etiology of this process iscontroversial. To evaluate whether alterations in the structure of thepulmonary capillary bed or the peribronchiolar arterioles could becorrelated with the pulmonary arterial pressure (Ppa), we examined thepulmonary vasculature in guinea pigs that had developed pulmonaryhypertension after being exposed to cigarette smoke for 6 mo. Thesmoke-exposed animals had a significant increased Ppa compared with thecontrol (air-exposed) animals (14.4 ± 2.4 vs. 9.9 ± 0.9 cmH₂O). In the smoke-exposedanimals, there was an increased percentage of muscularized peribronchiolar arterioles (33.5 ± 5.8% smoke exposed vs. 56.1 ± 5.8% control), and the capillary diameter and density weresignificantly decreased in both the center and periphery of the lobule(center diameter 8.8 ± 1.9, periphery diameter 10.0 ± 2.0 µm,center density 79 ± 5, and periphery density 84 ± 4 in smokedexposed vs. center diameter 7.7 ± 1.9, periphery diameter 8.6 ± 2.0 µm, center density 73 ± 6, and periphery density 77 ± 6 in controls). Neither group showed any correlation betweenthese values and the Ppa. We conclude that although chronic exposure tocigarette smoke produces alteration of the capillary bed and pulmonaryarterioles secondary to emphysematous air-space enlargement, thesestructural findings cannot explain the increase in Ppa. It appears thatpulmonary hypertension due to chronic cigarette smoke exposure is aresult of a primary alteration of capillary or muscular arteriolarvascular structure but instead may be secondary to alterations of thedynamic properties of the vascular bed with subsequent increase invascular resistance.

     

Exercise training enhances adrenergic constriction and dilation in the rat spinotrapezius muscle

Treadmill training increases functionalvasodilation in the rat spinotrapezius muscle, although there is noacute increase in blood flow and no increase in oxidative capacity. Toassess concurrent changes in vascular reactivity, we measured arterial diameters in the spinotrapezius muscle of sedentary (Sed) and treadmill-trained (Tr; 9-10 wk; terminal intensity 30 m/min,1.5° incline, for 90 min) rats during iontophoretic application of norepinephrine, epinephrine (Epi), andH⁺ (HCl) and during superfusionwith adenosine. Terminal-feed arteries and first-order arterioles in Trrats constricted more than those in Sed rats at the higher currentdoses of norepinephrine and Epi. In contrast, at low-current doses ofEpi, first- and second-order arterioles dilated in Tr but not in Sedrats. The vascular responses to HCl were highly variable, butsecond-order arterioles of Tr rats constricted more than those of Sedrats at intermediate-current doses. There were no significantdifferences between Sed and Tr rats in the vascular responses toadenosine. Both adrenergic vasodilation and vasoconstriction wereenhanced in the spinotrapezius muscle of Tr rats, and enhancedadrenergic vasodilation may contribute to increased functionalvasodilation. These observations further demonstrate vascularadaptations in "nontrained" skeletal muscle tissues.

     

Anatomic distribution of pulmonary vascular compliance

Presson, Robert G., Jr., Said H. Audi, Christopher C. Hanger, Gerald M. Zenk, Richard A. Sidner, John H. Linehan, Wiltz W. Wagner, Jr., and Christopher A. Dawson. Anatomic distribution ofpulmonary vascular compliance. J. Appl.Physiol. 84(1): 303-310, 1998.Previously, thepressure changes after arterial and venous occlusion have been used tocharacterize the longitudinal distribution of pulmonary vascularresistance with respect to vascular compliance using compartmentalmodels. However, the compartments have not been defined anatomically.Using video microscopy of the subpleural microcirculation, we havemeasured the flow changes in ~40-µm arterioles and venules aftervenous, arterial, and double occlusion maneuvers. The quasi-steadyflows through these vessels after venous occlusion permitted anestimation of the compliance in three anatomic segments: arteries >40µm, veins >40 µm, and vessels <40 µm in diameter. We foundthat ~65% of the total pulmonary vascular compliance was in vessels<40 µm, presumably mostly capillaries. The transient portions ofthe pressure and flow data after venous, arterial, and double occlusionwere consistent with most of the arterial compliance being upstreamfrom most of the arterial resistance and most of the venous compliancebeing downstream from most of the venous resistance.

     

Assessment of regional deposition of inhaled particles in human lungs by serial bolus delivery method

Kim, Chong S., S. C. Hu, P. DeWitt, and T. R. Gerrity.Assessment of regional deposition of inhaled particles in human lungs by serial bolus delivery method. J. Appl.Physiol. 81(5): 2203-2213, 1996.Detailedregional deposition of inhaled particles was investigated in youngadults (n = 11) by use of aserial bolus aerosol delivery technique. A small bolus (45 mlhalf-width) of monodisperse aerosols [1-, 3-, and5-µm particle diameter(D_p)] wasdelivered sequentially to a specific volumetric depth of the lung(100-500 ml in 50-ml increments), while the subject inhaled cleanair via a laser aerosol photometer (25-ml dead volume) with a constantflow rate ( = 150, 250, and 500 ml/s) andexhaled with the same without a pause to theresidual volume. Deposition efficiency (LDE) and deposition fraction in10 local volumetric regions and total deposition fraction of the lungwere obtained. LDE increased monotonically with increasing lung depthfor all three D_p.LDE was greater with smaller values in all lungregions. Deposition was distributed fairly evenly throughout the lungregions with a tendency for an enhancement in the distal lung regions for D_p = 1 µm.Deposition distribution was highly uneven forD_p = 3 and 5 µm, and the region of the peak deposition shifted toward the proximalregions with increasingD_p. Surface dosewas 1-5 times greater in the small airway regions and 2-17times greater in the large airway regions than in the alveolar regions.The results suggest that local or regional enhancement of deposition occurs in healthy subjects and that the local enhancement can be animportant factor in health risk assessment of inhaled particles.

     

Effects of endurance training on the cardiovascular system and water compartments in elderly subjects

Pickering, Gisèle P., Nicole Fellmann, BéatriceMorio, Patrick Ritz, Aimé Amonchot, Michel Vermorel, and JeanCoudert. Effects of endurance training on the cardiovascularsystem and water compartments in elderly subjects. J. Appl. Physiol. 83(4): 1300-1306, 1997.Theeffects of endurance training on the water compartments and thecardiovascular system were determined in 10 elderly subjects [age62 ± 2 yr, pretraining maximal oxygen consumption(O_{2 max})/kg = 25 ± 2 ml · min¹ · kg¹body wt]. They trained on a cycloergometer 3 times/wk for 16 wk(50-80%O_{2 max},then 80-85%O_{2 max}). They werechecked at 8 wk, 16 wk, and 4 mo after detraining. Training improvedO_{2 max} (+16%) andinduced plasma volume expansion (+11%). No change in total body water,extracellular fluid, interstitial and intracellular fluid volumes,fat-free mass, and body weight was detected in this small sample withtraining. Body fat mass decreased (2.1 ± 2.2 kg).Echocardiography at rest showed increased fractional shortening andejection fraction and decreased left ventricular end-systolic dimension(P < 0.05). Blood volume expansioncorrelates with cardiac contractility and has an impact on cardiacfunction. These improvements are precarious, however, and arecompletely lost after 4 mo of detraining, when elderly subjects losethe constraints and the social stimulation of the imposed protocol.

     

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization ofagonist-induced Ca²⁺ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca²⁺ concentration([Ca²⁺]_i)in peripheral cytoplasmic processes from where activation propagated asa[Ca²⁺]_iwave toward the central regions of the cell. The average propagation velocity of the[Ca²⁺]_iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca²⁺]_idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca²⁺]_ideclined monotonically, whereas in the central region[Ca²⁺]_idecreased in an oscillatory fashion. Propagating[Ca²⁺]_iwaves were preceded by small, highly localized[Ca²⁺]_itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa²⁺ release was 23 nM, and theunderlying flux of Ca²⁺ amountedto ~1-2 × 10¹⁸mol/s or ~0.3 pA, consistent with aCa²⁺ flux through a single orsmall number of endoplasmic reticulum Ca²⁺-release channels.

     

Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs

To determine theinitial signaling event in the vascular permeability increase afterhigh airway pressure injury, we compared groups of lungs ventilated atdifferent peak inflation pressures (PIPs) with (gadolinium group) andwithout (control group) infusion of 20 µM gadolinium chloride, aninhibitor of endothelial stretch-activated cationchannels. Microvascular permeability was assessed by using the capillary filtration coefficient(K_fc), ameasure of capillary hydraulic conductivity.K_fc was measuredafter ventilation for 30-min periods with 7, 20, and 30 cmH₂O PIP with 3 cmH₂O positive end-expiratorypressure and with 35 cmH₂O PIPwith 8 cmH₂O positive end-expiratory pressure. In control lungs,K_fc increasedsignificantly to 1.8 and 3.7 times baseline after 30 and 35 cmH₂O PIP, respectively. In thegadolinium group,K_fc was unchangedfrom baseline (0.060 ± 0.010 ml · min¹ · cmH₂O¹ · 100 g¹) after any PIPventilation period. Pulmonary vascular resistance increasedsignificantly from baseline in both groups before the lastK_fc measurementbut was not different between groups. These results suggest thatmicrovascular permeability is actively modulated by a cellular responseto mechanical injury and that stretch-activated cation channels mayinitiate this response through increases in intracellular calciumconcentration.

     

Catecholaminergic modulation of respiratory rhythm in an in vitro turtle brain stem preparation

An in vitro brainstem preparation from adult turtles was used to determine effects ofdopamine (DA) and norepinephrine (NE) on the pattern of respiratorymotor output recorded from hypoglossal nerve roots (XII). Bath-appliedDA (10-200 µM) increased the frequency of respiratory bursts(peaks) from 0.9 ± 0.2 to 2.4 ± 0.3 (SE) peaks/min, resultingin a 99 ± 9% increase in neural minute activity. R[+]-SCH-23390 (10 µM,D₁ antagonist) and eticlopride (20 µM, D₂ antagonist) attenuatedthe DA-mediated increase in peak frequency by 52 and 59%,respectively. On the other hand, the DA-receptor agonists apomorphine(D₁,D₂), quinelorane(D₂), and SKF-38393 (D₁) had no effect on peakfrequency. Prazosin, an₁-adrenergic antagonist (250 nM) abolished the DA-mediated frequency increase. Although NE(10-200 µM) and phenylephrine (10-200 µM,₁-adrenergic agonist) increasedpeak frequency from 0.5 ± 0.1 to 1.2 ± 0.3 peaks/min and from0.6 ± 0.1 to 1.0 ± 0.2 peaks/min, respectively, these effectswere not as large as that with DA alone. The data suggest that bothdopaminergic and adrenergic receptor activation in the brain stemincrease respiratory frequency in turtles, but the DA receptor-mediatedincrease is dependent on coactivation of₁-adrenergic receptors.

     

Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions

Phosphatidylinositol4,5-bisphosphate (PIP₂) affects profoundly several cardiacion channels and transporters, and studies ofPIP₂-sensitive currents in excised patches suggest thatPIP₂ can be synthesized and broken down within 30 s.To test when, and if, total phosphatidylinositol 4-phosphate (PIP) andPIP₂ levels actually change in intact heart, we used a new,nonradioactive HPLC method to quantify anionic phospholipids. Total PIPand PIP₂ levels (10-30 µmol/kg wet weight) do notchange, or even increase, with activation ofG_q/phospholipase C (PLC)-dependent pathways by carbachol(50 µM), phenylephrine (50 µM), and endothelin-1 (0.3 µM).Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1µM) bothcause 30% reduction of PIP₂ in ventricles, suggesting thatdiacylglycerol (DAG)-dependent mechanisms negatively regulate cardiacPIP₂. PIP₂, but not PIP, increases reversiblyby 30% during electrical stimulation (2 Hz for 5 min) in guinea pigleft atria; the increase is blocked by nickel (2 mM). Both PIP andPIP₂ increase within 3 min in hypertonic solutions, roughlyin proportion to osmolarity, and similar effects occur in multiple celllines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP₂ metabolismmight be sensitive to membrane tension, per se.

     

Dopamine, sensory discharge, and stimulus interaction with CO2 and O2 in cat carotid body

It is hypothesized that carotid bodychemosensory activity is coupled to neurosecretion. The purpose of thisstudy was to examine whether there was a correspondence between carotidbody tissue dopamine (DA) levels and neuronal discharge (ND) measured from the carotid sinus nerve of perfused cat carotid bodies and tocharacterize interaction betweenCO₂ andO₂ in these responses. ND andtissue DA were measured after changing from normoxic, normocapnic control bicarbonate buffer (PO₂>120 Torr, PCO₂ 25-30 Torr, pH ~ 7.4) to normoxic hypercapnia(PCO₂ 55-57 Torr, pH7.1-7.2) or to hypoxic solutions(PO₂ 30-35 Torr) withnormocapnia (PCO₂ 25-30 Torr, pH ~ 7.4) or hypocapnia (PCO₂10-15 Torr, pH 7.6-7.8). Similar temporal changes for ND and tissue DA were found for all of the stimuli, although there was a much different proportional relationship fornormoxic hypercapnia. Both ND and DA increased above baseline valuesduring flow interruption and normocapnic hypoxia, and both decreasedbelow baseline values during hypoxic hypocapnia. In contrast, normoxichypercapnia caused an initial increase in ND, from a baseline of 175 ± 12 (SE) to a peak of 593 ± 20 impulses/s within 4.6 ± 0.9 s, followed by adaptation, whereas ND declined to 423 ± 20 impulses/s after 1 min. Tissue DA initially increased from a baselineof 17.9 ± 1.2 µM to a peak of 23.2 ± 1.2 µM within 3.0 ± 0.7 s, then declined to 2.6 ± 1.0 µM. The substantialdecrease in tissue DA during normoxic hypercapnia was not consistentwith the parallel changes in DA with ND that were observed for hypoxic stimuli.

     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

Paradoxical helium and sulfur hexafluoride single-breath washouts in short-term vs. sustained microgravity

Lauzon, Anne-Marie, G. Kim Prisk, Ann R. Elliott, SylviaVerbanck, Manuel Paiva, and John B. West. Paradoxical helium andsulfur hexafluoride single-breath washouts in short-term vs. sustainedmicrogravity. J. Appl. Physiol. 82(3):859-865, 1997.During single-breath washouts in normal gravity (1 G), the phase III slope of sulfur hexafluoride(SF₆) is steeper than that ofhelium (He). Two mechanisms can account for this:1) the higher diffusivity of Heenhances its homogeneous distribution; and2) the lower diffusivity ofSF₆ results in a more peripherallocation of the diffusion front, where airway asymmetry is larger.These mechanisms were thought to be gravity independent. However, weshowed during the Spacelab Life Sciences-2 spaceflight that insustained microgravity (µG) theSF₆-to-He slope difference isabolished. We repeated the protocol during short periods (27 s) of µG(parabolic flights). The subjects performed a vital-capacityinspiration and expiration of a gas containing 5% He-1.25%SF₆-balanceO₂. As in sustained µG, thephase III slopes of He and SF₆decreased. However, during short-term µG, theSF₆-to-He slope differenceincreased from 0.17 ± 0.03%/l in 1 G to 0.29 ± 0.06%/l inµG, respectively. This is contrary to sustained µG, in which theSF₆-to-He slope difference decreased from 0.25 ± 0.03%/l in 1 G to 0.01 ± 0.06%/lin µG. The increase in phase III slope difference in short-term µGwas caused by a larger decrease of He phase III slope compared with that in sustained µG. This suggests that changes in peripheral gasmixing seen in sustained µG are mainly due to alterations in thediffusive-convective inhomogeneity of He that require >27 s of µGto occur. Changes in pulmonary blood volume distribution or cardiogenicmixing may explain the differences between the results found inshort-term and sustained µG.

     

Vasoconstrictor responses of coronary resistance arteries in exercise-trained pigs

Coronaryresistance arteries isolated from exercise-trained pigs have been shownto exhibit enhanced myogenic reactivity (J. M. Muller, P. R. Myers, andM. Harold Laughlin. J. Appl. Physiol. 75: 2677-2682, 1993). The purpose of this study was to test the hypothesis that exercise training results in enhanced vasoconstrictor responses of these arteries to all vasoconstrictor stimuli[specifically acetylcholine (ACh), endothelin-1 (ET-1), KCl, andthe Ca²⁺ channel-agonist Bay K8644]. Female Yucatan miniature swine were trained (Trn) on amotor-driven treadmill (n = 16) orremained sedentary (Sed, n = 15) for16-20 wk. Arteries 50-120 µm in diameter were isolated andcannulated with micropipettes, and intraluminal pressure was set at 60 cmH₂O throughout experiments.Vasoreactivity was evaluated by examining constrictor responses toincreasing concentrations of ACh(10⁹ to10⁴ M), ET-1(10¹⁰ to10⁸ M), KCl (bathreplacement with isotonic physiological saline solution containing 30 or 80 mM), and Bay K 8644 (10⁹ to10⁶ M). Constricteddiameters are expressed relative to the passive diameter observed after100 µM SNP. All four constrictors produced similar decreases indiameter in arteries from both groups [ACh: 0.52 ± 0.07 (Trn)and 0.54 ± 0,06 (Sed); ET-1: 0.66 ± 0.05 (Trn) and 0.70 ± 0.07 (Sed); KCl: 0.66 ± 0.05 (Trn) and 0.70 ± 0.07 (Sed); Bay K8644: 0.86 ± 0.05 (Trn) and 0.76 ± 0.05 (Sed)]. Present results combined with previous observations indicate that exercise training does not alter vasoconstrictor responses of porcine coronary resistance arteries but specifically increases myogenic reactivity. Thus the underlying cellular mechanisms for myogenic tone are alteredby training but not receptor-mediated mechanisms (ACh and ET-1) norvoltage-gated Ca²⁺ channels (KCland Bay K 8644) in coronary resistance arteries.

     

Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat

The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca²⁺]_i andCa²⁺ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca²⁺), the resting length and[Ca²⁺]_i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 10⁵ M) caused transient increasein [Ca²⁺]_i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca²⁺]_i transient was notsignificantly different, but the maintained [Ca²⁺]_i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa²⁺-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca²⁺]_i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca²⁺]_i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca²⁺]_i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca²⁺]_i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca²⁺]_i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca²⁺]_i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (10⁸ M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca²⁺]_i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca²⁺]_i and contraction of vascularsmooth muscle triggered by Ca²⁺ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca²⁺]_i due to Ca²⁺release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca²⁺]_i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

     

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells

These experiments were performed totest the hypotheses that myosin light chain 17 (MLC₁₇) aand b isoform expression varies between individual vascular smoothmuscle (SM) cells and that their expression correlates with cellunloaded shortening velocity. Single SM cells isolated from rabbitaorta and carotid arteries were used to measure unloaded shorteningvelocity and subsequently were analyzed via RT-PCR forMLC₁₇ a and b mRNA ratio. The MLC_17b/a mRNA andprotein ratios from adjacent tissue sections correlate very well(R² = 0.68), allowing use of the mRNA ratio topredict the protein ratio. The rabbit MLC₁₇ isoform proteinsequence was found to be similar to, but unique from, the swine, mouse,and chicken sequences. Isolated single SM cells from the aorta andcarotid have resting lengths of 70-280 µm and shorten to33-88 µm after contraction. Isolated cell maximum unloadedshortening velocity is highly variable (0.5-7.5 µm/s) butbecomes more uniform when normalized to initial cell length(0.01-0.05 cell lengths/s). Carotid cells activated in thepresence of okadaic acid (1 µm) have mean maximal unloaded shorteningvelocities not significantly different from carotid cells activatedwithout okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting celllength before activation is significantly correlated with final celllength after unloaded shortening. Neither initial cell length, finalcell length, total cell length change, nor maximum unloaded shorteningvelocity (absolute or normalized) was significantly correlated withsingle-cell MLC_17b/a mRNA ratio. These studies wereperformed in isolated single SM cells where unloaded shorteningvelocity and MLC_17b/a mRNA ratios were measured in the samecell. In this preparation, the three-dimensional organization andmilieu of the cell is kept intact, but without the intercellularheterogeneity concerns of multicellular preparations. These resultssuggest the MLC_17b/a ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloadedshortening velocity in single SM cells.

     

Regional deposition of inhaled particles in human lungs: comparison between men and women

We measured detailed regional depositionpatterns of inhaled particles in healthy adult male(n = 11; 25 ± 4 yr of age) and female (n = 11; 25 ± 3 yr of age)subjects by means of a serial bolus aerosol delivery technique formonodisperse fine [particle diameter(D_p) = 1 µm] and coarse aerosols(D_p = 3 and 5 µm). The bolus aerosol (40 ml half-width) was delivered to a specificvolumetric depth (Vp) of the lung ranging from 100 to 500 ml with a50-ml increment, and local deposition fraction (LDF) was assessed for each of the 10 local volumetric regions. In all subjects, the deposition distribution pattern was very uneven with respect to Vp,showing characteristic unimodal curves with respect to particle sizeand flow rate. However, the unevenness was more pronounced in women.LDF tended to be greater in all regions of the lung in women than inmen for D_p = 1 µm. For D_p = 3 and 5 µm, LDF showed a marked enhancement in the shallow region of Vp  200 ml in women compared with men(P < 0.05). LDF in women wascomparable to or smaller than those of men in deep lung regions of Vp > 200 ml. Total lung deposition was comparable between men and womenfor fine particles but was consistently greater in women than men forcoarse particles regardless of flow rates used: the difference rangedfrom 9 to 31% and was greater with higher flow rates(P < 0.05). The results indicatethat 1) particledeposition characteristics differ between healthy men and women undercontrolled breathing conditions and2) deposition in women is greaterthan that in men.

     

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

It has been reported thatsecretory mammary epithelial cells (MEC) release ATP, UTP, and UDP uponmechanical stimulation. Here we examined the physiological changescaused by ATP/UTP in nontransformed, clonal mouse mammary epithelia(31EG4 cells). In control conditions, transepithelial potential (apicalside negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4  · cm², respectively. The apicalmembrane potential was 43.9 ± 1.7 mV, and the ratio of apicalto basolateral membrane resistance (R_A/R_B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranescaused large voltage and resistance changes with an EC₅₀ of~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreasedR_A/R_B by a factor of3. The addition of adenosine to either side (100 µM) hadno effect on any of these parameters. The ATP/UTP responses werepartially inhibited by DIDS and suramin and mediated by a transientincrease in free intracellular Ca²⁺ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca²⁺ increase was blocked by cyclopiazonic acid, by BAPTA,or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbedfluid in the resting state, and ATP or UTP increased fluid secretion by5.6 ± 3 µl · cm² · h¹(n = 10). Pharmacology experiments indicate that 31EG4epithelia contain P2Y₂ purinoceptors on the apical andbasolateral membranes, which upon activation stimulate apicalCa²⁺-dependent Cl channels and cause fluid secretion acrossthe monolayer. This suggests that extracellular nucleotides could playa fundamental role in mammary gland paracrine signaling and theregulation of milk composition in vivo.