Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

Uterine venous permeability in the rat is altered in response to pregnancy, vascular endothelial growth factor, and venous constriction.

Venoarterial communication is a potential short-loop signaling pathway for the local control of uteroplacental perfusion. As this pathway is permeability-dependent, this study investigated the effects of molecular weight, gestation, vascular endothelial growth factor (VEGF), wall tension, and constriction on solute flux across the venous wall. Experiments utilized fluorimetry to quantitate solute flux (3- and 70-kDa dextran) in isolated segments of uterine vein from virgin (NP) and late-pregnant (LP; day 20) rats as a function of endothelial surface area and time. Uterine veins were > 10-fold more permeable to the 3- versus 70-kDa dextran in both NP and LP groups. Flux was increased during gestation ( 2.5-fold), and by VEGF (NP, 3 kDa: 3.3-fold, 70 kDa: 4.8-fold; LP, 3-kDa: 3.3-fold, 70 kDa: 7.4-fold). Permeability to the 3 kDa dextran correlated directly with wall tension (r2 = .74 in both groups), whereas permeability to both dextrans correlated inversely with constriction (NP: r2 = .85 and .76; LP: r2 = .89 and .79, respectively). Uterine veins demonstrate permeability to 3- and 70-kDa tracers resulting from molecular weight dependence and an apparent difference in transport mechanisms. Permeability is enhanced by gestational remodeling and subject to modulation by placental factors, indicating the presence of a regulated pathway for the transfer of molecules across the venous wall.

Differential expression and regulation of the vascular endothelial growth factor receptors neuropilin-1 and neuropilin-2 in rat uterus.

Vascular endothelial growth factor (VEGF) is a potent modulator of vascular remodeling and angiogenesis in the uterus. Recently, neuropilins (Npn), semaphorin receptors associated with neuronal guidance, were demonstrated to bind VEGF isoforms with high affinity, facilitating VEGF(165) binding to the tyrosine kinase receptor VEGFR2. The current studies examined rat uterus neuropilin expression and regulation. Npn-1 and Npn-2 transcripts and 135-kDa proteins were observed in uterine extracts. Both uterine vascular endothelial cells and glandular epithelium expressed Npn-1 immunoreactivity, whereas Npn-2 was restricted to the glandular epithelium. In hormone-replaced ovariectomized animals, progesterone increased uterine 6.5-kb Npn-1 messenger RNA (mRNA) expression approximately 2-fold compared with that in tissues from ovariectomized controls. 17ss-Estradiol alone had no effect, but blunted the progesterone response; by contrast, Npn-2 mRNA expression was decreased by estrogen. VEGFR2 mRNA was coregulated with Npn-1. Consistent with these results, Npn-1 mRNA expression was augmented nearly 7- and 4-fold at metestrus and diestrus, respectively, during periods of high progesterone; Npn-2 mRNA expression was not significantly altered during the estrous cycle. The regulated expression and differential localization of neuropilins in the rat uterus suggest that these receptors may participate in hormonally regulated changes occurring throughout the female reproductive cycle.

Synthetic atrial natriuretic factor does not dilate resistance-sized arteries.

The effects of synthetic atrial natriuretic factor and atriopeptin III on induced tone in resistance-sized arteries from the rat were examined in vitro. Cylindrical segments of small mesenteric or cerebral arteries were mounted on a microcannula and pressurized to a transmural pressure of 75 mm Hg. After equilibration, the level of tone in cerebral arteries was on the order of - 35 change in diameter; addition of atrial natriuretic factor or atriopeptin III in cumulative doses from 10(-10) to 10(-7) M did not produce any transient or sustained changes in diameter. Similarly, atrial natriuretic factor or atriopeptin III did not alter the contractile responses of cerebral vessels to serotonin or prostaglandin F2 alpha. Mesenteric arteries, which do not possess an intrinsic myogenic tone, were precontracted with potassium (30 mM), norepinephrine (10(-6) M), or prostaglandin F2 alpha (1.1 X 10(-5) M) and exposed to the synthetic natriuretic peptides, also without effect. Transmural electrical stimulation (0.3-msec pulses; 180 mA; 4/second) relaxed cerebral and contracted mesenteric arteries; preincubation in 10(-7) M atrial natriuretic factor or atriopeptin III did not alter subsequent responses. These observations suggest that the hypotensive action of atrial natriuretic factor cannot be attributed to direct vasodilation of splanchnic or cerebral resistance-sized arteries.

The effects of photoperiod and lid suture on eye growth in chickens.

Three models of postnatal eye enlargement in leghorn chicks (surgical fusion of the eyelids, MES; exposure to continuous light, 24L; prolonged darkness, OL) were characterized on a morphologic and temporal basis, and a relationship between photoperiod and eye growth was described. While pronounced enlargement of the eye was evident in the OL and MES groups after 6 wk, a 15-wk experimental period was necessary to produce significant (P less than 0.05) eye enlargement under 24L. This enlargement was unaffected by pinealectomy and was characterized by increased equatorial diameter and decreased axial length. The macrophthalmos resulting from both MES and OL was characterized by increases in absolute and relative eye weights, axial length, and equatorial diameter. The MES eyes, however, showed a pronounced bulging of the cornea and increased anterior chamber depth and equatorial diameter, while those from the OL group had a flattened cornea and decreased anterior chamber depth. Finally, a relationship between photoperiod and eye growth was established and was best described by an inverse, continuous semilogarithmic function.

Estrogen replacement modulates resistance artery smooth muscle and endothelial alpha2-adrenoceptor reactivity.

The purpose of this study was to evaluate the effects of estrogen replacement on ovariectomized rats on the reactivity to alpha2-adrenoceptor activation, and to analyze the role of the endothelium in modulating this response. Third order branches of the superior mesenteric artery from ovariectomized untreated (OvX) and estrogen-replaced (E2) Sprague-Dawley rats were cannulated and pressurized to 50 mmHg. Under relaxed conditions (0.1 mM papaverine), there were no differences in lumen diameter. Intact vessels from E2 rats were unresponsive to clonidine (0.01-10 microM); incubation in indomethacin (1 microM), a cyclooxygenase inhibitor, produced intermediate constriction that was significantly augmented by L-NNA (0.3 mM), a NO synthase inhibitor, or by endothelial denudation. Conversely, intact vessels from OvX animals constricted to clonidine in a concentration-dependent manner. This effect was significantly diminished by endothelial removal or indomethacin, but was not affected by L-NNA. Yohimbine (1 microM), an beta2 receptor antagonist, significantly diminished arterial sensitivity to, and efficacy of clonidine. These results suggest that estrogen replacement enhanced vasoconstriction induced by smooth muscle alpha2 adrenoceptor activation, although this effect was obscured in intact vessels due to an overriding influence of endothelial dilator substances, primarily NO. In arteries from OvX animals, smooth muscle was less sensitive to alpha2 agonist stimulation, however, the release of a vasoconstrictor prostanoid from the endothelium was predominant, and induced significant vasoconstriction.

Intolerance to volume expansion: a theorized mechanism for the development of preeclampsia.

We present a theorized mechanism for the development of preeclampsia, suggesting that one important underlying pathophysiologic mechanism is intolerance to volume expansion. The stage is set for this intolerance by chronic volume constriction, which leads to a requirement for increased basal peripheral vasoconstrictor tone to maintain blood pressure and allow for continued perfusion of the upright hominid head. In pregnancy, volume expansion signaled by the placenta cannot be accommodated by the constricted vascular system. The inability of the normally adaptive endothelial vasodilatory mechanisms to overcome the chronic vasoconstrictor tone leads to endothelial damage, exacerbation of vasoconstriction, and clinical hypertension. Disease resolution, characterized by diuresis, occurs with the elimination of the placenta-derived drive to retain volume. The reason preeclampsia does not recur uniformly with subsequent pregnancy is permanent restructuring of the maternal cardiovascular system with pregnancy that allows for greater plasma volume expansion in future gestations.

Flow decreases myogenic reactivity of mesenteric arteries from pregnant rats.

OBJECTIVE: To determine whether pregnancy alters the response of small mesenteric arteries to increased pressure (myogenic reactivity) and flow. METHODS: Mesenteric arteries (300 microns) from cycling nonpregnant (NP, n = 6) and late pregnant (20 days, LP, n = 6) Sprague-Dawley rats were dissected and mounted on an arteriograph system designed for the precise measurement of pressure and flow. Myogenic reactivity was measured as the percentage constriction after a pressure increase to 75 mmHg in the absence and presence of flow (60 microL/minute). RESULTS: In the absence of flow, there was no difference in myogenic reactivity in arteries from NP versus LP animals (NP, 8.4 +/- 1.4%; LP, 11.0 +/- 1.6%; not significant). In the presence of flow, myogenic reactivity was decreased in arteries from LP rats, but was unchanged in arteries from NP rats (NP, 13.2 +/- 1.1%; LP, 2.5 +/- 2.9%; P < .05). The differential group effect appeared to result not from differences in arterial response to changes in pressure or flow alone, but rather from the interaction between pressure and flow. CONCLUSION: These results suggest that pregnancy alters the interaction of the physical forces of pressure and flow on the arterial wall in a manner consistent with decreased vascular resistance.

A field study of the fertility of transported equine semen.

A field trial of artificial insemination in horses with transproted, chilled semen was conducted using a specially designed container which permitted a controlled, slow initial rate of cooling (-0.3 degrees C/min) and maintenance of a final temperature of 4 degrees -6 degrees C for more than 36 hrs. Forty-six mares in 23 states were inseminated with semen from three German Warmblood stallions standing at stud in Hamilton, Massachusetts. A third-cycle conception rate of 91% was obtained.

Effect of antihypertensive treatment on myogenic properties of brain arteries from the stroke-prone rat.

Using an in vitro pressurized technique, we measured the myogenic pressure range and degree of myogenic activity in posterior cerebral arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and rats of the same strain maintained by antihypertensive therapy. Treatment lowered the upper myogenic pressure limit and increased the strength of the myogenic response to levels comparable with those previously found in normotensive (Wistar-Kyoto, WKY) rats, suggesting that blood pressure history is the primary determinant of myogenic properties in the cerebral circulation.

Pre-existing level of tone is an important determinant of cerebral artery autoregulatory responsiveness.

The objective of this study was to test the hypothesis that cerebral artery autoregulatory responses to increasing transmural pressure depend on the pre-existing level of vascular tone. Pial arteries were obtained from adult normotensive Wistar-Kyoto (WKY) rats, and were cannulated and studied under pressurized conditions using a video-assisted perfusion system. Myogenic tone developed spontaneously during equilibration, reducing the lumen diameter by 31% (n = 6). Each artery was then subjected to a series of step increases in transmural pressure of between 50 and 100 mmHg, a manoeuvre that produces autoregulatory constriction. Afterwards, serotonin was added to the perfusate to decrease lumen diameter by another 27% and the transmural pressure changes were repeated once more. The autoregulatory effectiveness in response to a change of 50----100 mmHg in transmural pressure was expressed as both the percentage change in diameter and the myogenic 'gain'. During the increased activation induced by serotonin, the autoregulatory responses to a change in transmural pressure were completely abolished; lumen diameter, which had previously decreased by 4.9 +/- 1.5%, increased by 10.5 +/- 4.5% (P less than 0.05). The nature of this change is further reflected in the myogenic gain, which was 0.39 +/- 0.14 in vessels with myogenic tone alone, and -1.14 +/- 0.56 for the same arteries at the higher level of activation (P less than 0.05). In additional experiments (n = 6), a comparable degree of constriction was induced with 0.5% ethanol, which increases calcium influx through receptor-independent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturation is associated with changes in rat cerebral artery structure, biomechanical properties and tone.

AIM: This study evaluated the hypothesis that physiological maturation affects cerebral artery smooth muscle-endothelial interactions involved in pressure-induced tone and alters the dimensional and biomechanical properties of small posterior cerebral arteries (PCA). METHODS: Secondary branches of PCA from young (4-5 weeks old, n=11), adult (14-16 weeks old, n=11) and mature (44-47 weeks old, n=11) male Sprague-Dawley rats were isolated, cannulated, pressurized and subjected to a range of intraluminal pressures (10-110 mmHg) to determine tone with and without pharmacologic nitric oxide synthase (NOS) inhibition. Measurements of passive lumen diameter and wall thickness as a function of pressure were used to determine changes in structure, distensibility and wall stress; histological analysis was performed on vessel cross-sections to assess collagen and elastin contents. RESULTS: Although pressure-dependent tone decreased significantly during ageing, differences between groups were abolished by NOS inhibition. Vessel diameters increased in adult vs. young rats (at 90 mmHg, 233 ± 6.0 µm vs. 192 ± 4.5 µm; P<0.05), possibly secondary to somatic growth. Further ageing was associated with reductions in lumen diameter (207 ± 6.5 µm; P<0.05), increased wall and media thickness (and wall/lumen ratio) and cross-sectional area. Distensibility and wall collagen were unchanged, although elastin content was significantly reduced. CONCLUSIONS: Maturation is associated with differences in PCA dimensional properties that indicate a pattern of initial outward eutrophic, followed by inward hypertrophic remodelling. Functionally, the contribution of basal NO increases with age in a way that reduces pressure-dependent tone and diminishes vasodilator reserve.

Effects of etonogestrel treatment in the reproductive organs and uterine arteries of nonoophorectomized guinea pigs.

The endometria of women treated with long-term progestin-only contraceptives (LTPOCs) display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow, oxidative stress, and unpredictable focal abnormal endometrial bleeding. Because human studies on the effects of LTPOC treatment are constrained for ethical and practical reasons, we assessed the suitability of nonoophorectomized guinea pigs (GPs) to best mimic the hormonal milieu of women. The present study demonstrates that treatment of GPs parallels the morphological changes following LTPOC treatment of the human endometrium and ovaries. Specifically, treatment resulted in larger hyperemic, uteri compared with controls. Histopathologic and immunohistochemical analysis demonstrated fewer endometrial glands, decreased luminal mucus, increased numbers of blood vessels, and focal hemorrhage. While increased staining for the cell mitosis marker, Ki67, was present in the zona functionalis, no such increase occurred in the basalis. Lastly, effects on vasomotor features of uterine arteries suggest changes that favor increased resistance and reduced blood flow promoting decreased ability to withstand elevations in transmural pressure.

Mechanotransduction by vascular smooth muscle.

Mechanotransduction by vascular smooth muscle (VSM) is defined as a cellular response (contraction, secretion, growth, division) to transmural pressure or stretch. This review includes an overview of the physical forces VSM cells experience in vivo, consideration of experimental techniques used to study VSM mechanotransduction, and a discussion of the scientific literature pertinent to the individual cellular components that have been implicated in the transduction of physical forces. These include: the extracellular matrix, integrins, ion channels, the sarcoplasmic reticulum, second messenger systems, contractile proteins, and the cytoskeleton.

Spontaneous vasomotion in pressurized cerebral arteries from genetically hypertensive rats.

Resistance-sized branches of posterior cerebral arteries from Wistar-Kyoto (WKY), spontaneously hypertensive (SHR), spontaneously hypertensive stroke-prone (SHRSP), and antihypertensive-treated SHRSP (SHRSP-TRT) rats were studied in vitro. After the rats were killed, arterial segments were excised, mounted on microcannulas, and pressurized. After equilibration, intravascular pressure was increased in a stepwise fashion from 30 to 150-200 mmHg. All vessels developed a myogenic tone, which resulted in diameter reductions of 31-37% at 100 mmHg when compared with fully relaxed diameters [approximately 200 micron in 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. Differences in the extent of tone were not significant between animal groups (P greater than 0.05). Rhythmic vasomotion was present in 94% SHRSP and 100% SHRSP-TRT, 83% SHR, and only 6% of the WKY arteries. At higher pressures, the amplitude of the diameter oscillations decreased and frequency increased. Vasomotion was unaltered by tetrodotoxin or indomethacin, but could be abolished by cooling to 34 degrees C, ouabain (a depolarizing solution containing 125 mM K+), potassium-free physiological saline solution, or by calcium entry blockade with diltiazem or MnCl2. In normally quiescent WKY arteries, vasomotion, which was qualitatively similar to that observed in the hypertensive strains, could be induced by the addition of 5 mM tetraethylammonium chloride. Thus intrinsic oscillations in membrane calcium and potassium conductance may underlie the rhythmic contractile activity of rat cerebral arteries. This property appears to have a major genetic component, the expression of which is relatively independent of blood pressure history and is not related to the myogenic properties of the preparation.

Influence of transmural pressure of myogenic responses of isolated cerebral arteries of the rat.

We examined the diameter responses of isolated and pressurized posterior cerebral artery branches to various static and dynamic pressure alterations. These vessels, dissected from an anatomically identifiable location in the rat brain, developed tone when placed in a normal calcium physiological salt solution (1.6 mM Ca-PSS). Following a series of transmural pressure steps (delta p) of 25 or 50 mm Hg completed in 1-2 s and made every 5 min, they attained additional tone resulting in a mean luminal diameter of 139 micron at 100 mm Hg which was 35% less than their relaxed size measured in 1 mM EGTA-PSS. Continuous measurements of wall thickness and lumen diameter were obtained using a video electronic system in 1-2 mm long arterial segments, and autoregulatory gain factors calculated. Myogenic responses were obtained from each of 6 vessels taken from 6 WKY rats. Diameters following the step pressure changes were usually stable within 2-4 min. The data defined a myogenic regulatory pressure range from 49-145 mm Hg. Gain values averaged about 17% of that necessary for these arteries to maintain perfect flow autoregulation. Our results for myogenicity are comparable with the pressure range for blood flow autoregulation reported by others for the rat. We conclude that myogenic mechanisms, at least in this size artery, are partly responsible for flow autoregulation, and that they are supplemented by metabolic mechanisms operative in the intact rat brain.

Myogenic properties of cerebral blood vessels from normotensive and hypertensive rats.

Myogenic properties of posterior cerebral arteries from normotensive and hypertensive rats were analyzed in vitro and quantified in terms of both pressure range limits and degree of myogenic activity. Spontaneously hypertensive rat (SHR) vessels were significantly narrower in a fully relaxed state, and both wall thickness and wall-to-radius ratios were increased. After equilibration in 1.6 mM calcium physiological saline solution a substantial tone developed which resulted in average diameter decreases of 34 and 37% in Wistar-Kyoto (WKY) and SHR, respectively; average lumen diameters were approximately 125 micron. Rapid changes in transmural pressure (delta P 10-25 mmHg/s) were applied and diameter responses measured continuously. Myogenic responses began 1-3 s after a change in transmural pressure, and arteries regained their initial diameters after a pressure step in about 2 min; a final, steady-state diameter was achieved in 4-5 min. Myogenic pressure ranges were 49-145 mmHg in WKY and 64-181 in SHR; when responses were segregated according to positive and negative pressure steps, more myogenic responses were observed at lower pressures for pressure step decreases when compared with pressure step increases. Thus myogenic ranges for increasing pressure steps were 71-151 (WKY) and 72-188 mmHg (SHR) and for decreasing steps 45-117 (WKY) and 57-148 mmHg (SHR). Myogenic responses in SHR were weaker than in WKY rats: the former maintained essentially a constant diameter over a wide range of pressures, whereas arteries from the latter decreased diameter with increasing pressures.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance vessels in hypertension.

Hypertension differentially affects the functional behavior of the mesenteric resistance arteries compared with similar sized arteries from the cerebral vascular autoregulatory bed. Antihypertensive drug treatment may, or may not, reverse the vascular wall structural composition and smooth muscle cell receptor sensitivity and contractile properties. These effects depend on the agent used, the length of treatment, and the vascular bed. Rat brain resistance arteries are myogenic at transmural pressures that are similar to those measured in autoregulatory, in vivo flow studies, as is the elevation of this range as a consequence of hypertension. The methods we have developed afford the investigator quantitative control over the variables that might affect the function of resistance vessels when tested in vitro. These techniques maintain the vessel close to physiological, in vivo conditions, and are useful tools for unraveling the complex mechanisms that define both, the normal vessel properties, and those of the vessel which has undergone modifications as a result of hypertension. Whether the vascular changes that we find in the SHR rat model parallel those which occur in human essential hypertension must yet be ascertained.

Hypertrophic and hyperplastic effects of pregnancy on the rat uterine arterial wall.

OBJECTIVE: The purpose of this study was to characterize the cellular mechanisms (hypertrophy or hyperplasia) contributing to uterine arterial growth during pregnancy. STUDY DESIGN: Vascular smooth muscle cells were enzymatically dispersed from radial uterine arteries from nonpregnant (n = 4) and late pregnant (day 20 to 21, n = 6) Sprague-Dawley rats, and the axial length was measured. Midpregnant (days 9 to 11, n = 5), late pregnant (n = 5), and nonpregnant (n = 5) Sprague-Dawley rats were injected with bromodeoxyuridine, and immunohistochemistry was used to visualize dividing and nondividing vascular smooth muscle cells and endothelial cells in both radial and main uterine arteries. RESULTS: Vascular smooth muscle cells isolated from late-pregnant vessels were 21% longer than those from nonpregnant animals, increasing from 118 +/- 4 microns in nonpregnant rats to 150 +/- 7 microns in late-pregnant rats. Cell proliferation rates of both vascular smooth muscle cells and endothelial cells were quite low in nonpregnant animals but increased significantly during pregnancy in both radial and main uterine arteries. The pattern of changes in cell division rates over time, however, varied between the different arterial segments: in midpregnancy rates of both vascular smooth muscle cell and endothelial cell division were highest in the smaller radial arteries, whereas at term the mitotic index values were increased in main uterine arteries. CONCLUSIONS: Gestational growth of the uterine vasculature is accomplished by a combination of both cellular hypertrophy and hyperplasia in the arterial wall. Furthermore, local differences in cell division rates at different times during gestation suggest the existence of spatially discrete growth mechanisms within the vascular network.

Pregnancy-induced changes in the three-dimensional mechanical properties of pressurized rat uteroplacental (radial) arteries.

OBJECTIVE: The purpose of this study was to describe the effects of pregnancy on the size and three-dimensional mechanics of uterine radial arteries. STUDY DESIGN: Measurements of lumen diameter, wall thickness, and axial and radial distensibility were made in situ and in pressurized segments of excised vessels from nonpregnant (n = 29) and late-pregnant (days 19 to 21, n = 19) Sprague-Dawley rats. RESULTS: In the unpressurized state, the overall length of the radial artery segment of the arcade increased approximately 4.8 times during gestation. Lumen diameter increased by 60%, as did distensibility in both the radial and axial directions. However, there was no measurable change in the thickness of the vascular wall, which was muscular in appearance and comprised approximately two layers of circumferentially oriented smooth muscle, a relatively thick internal elastic lamina, and a well-defined endothelial layer. Cross-sectional area increased significantly during pregnancy (1.37 times at 50 mm Hg), as did the overall volume of the vascular wall (6.86 times at 50 mm Hg), primarily as a result of arterial growth in the longitudinal (axial) direction. CONCLUSIONS: Remodeling of the radial artery segment of the uterine vasculature clearly occurs during gestation, resulting in vessels that are of a larger caliber and are longer and more distensible in both the axial and radial directions.