Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 ± 3.6 to 110 ± 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 ± 8.9 to 76 ± 8.8 mmHg, N = 7) or pentobarbital (from 118 ± 4.5 to 57 ± 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 ± 4.1 to 92 ± 5.9 mmHg, N = 7) and anesthetized rats (from 123 ± 6.9 to 87 ± 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition

Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats.

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 +/- 3.6 to 110 +/- 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 +/- 8.9 to 76 +/- 8.8 mmHg, N = 7) or pentobarbital (from 118 +/- 4.5 to 57 +/- 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 +/- 4.1 to 92 +/- 5.9 mmHg, N = 7) and anesthetized rats (from 123 +/- 6.9 to 87 +/- 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition.

Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle.

Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.

Human cervical cancer. Retinoids, interferon and human papillomavirus.

Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16-immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways).

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells.

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3-20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 microM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI.

Modulation of phospholipase A2 activity and sodium transport by angiotensin-(1-7).

Angiotensin II (Ang II) receptors are coupled to a variety of signal transduction mechanisms. In the kidney, Ang II at nanomolar concentration binds to proximal tubular cells and stimulates phospholipase A2 (PLA2), which in turn catalyzes the hydrolysis of phosphatidylcholine into lysophosphatidylcholine (LPC) and fatty acid. This signal transduction pathway has been shown to be an important modulator of sodium transport. The kidney cortex possesses the enzyme necessary to convert angiotensin I (Ang I) directly to Ang-(1-7) bypassing Ang II as an intermediate. The present investigation was undertaken to determine whether Ang-(1-7) influences epithelial cell function by comparing this heptapeptide with Ang II as a modulator of PLA2 activity and sodium transport. Proximal tubular cells were labeled in tissue culture with 3H-choline and PLA2 activity was measured by quantitation of LPC. We found that Ang II (10(-9) M to 10(-6) M) significantly increased PLA2 activity (154 +/- 36% to 209 +/- 94%). Similar results were obtained with Ang-(1-7) (240 +/- 130% to 353 +/- 40%). The bioactivity of the peptides was assayed by its ability to regulate transcellular 22Na flux. Ang II (10(-9) M) inhibited 22Na flux by 12 +/- 2% while Ang-(1-7) (10(-9) M) inhibited 22Na flux by 20 +/- 5%. These results suggest that one potential role of Ang-(1-7) in the regulation of kidney epithelial electrolyte transport may involve activation of PLA2.

Delivery of growth factor to wounds using a genetically engineered biological bandage.

Increasing the rate of wound healing of acute wounds and promoting the closure of chronic ulcers is an important goal in wound therapy. Growth factors have been shown to facilitate this process; however, the systems described for growth factor delivery are not ideal. In the present report we demonstrate the feasibility of a new method of delivering growth factors to the wound site using a genetically engineered biological bandage. The bandage consists of keratinocytes (SCC-13 cells) that are engineered by gene transfer to produce high levels of bovine growth hormone (bGH). bGH was selected for these studies because it can be easily distinguished from rat and human growth hormone in wound fluids and culture medium. The bGH-producing cells are contained and maintained in serum-free medium inside an envelope composed of a low protein binding, 0.2 micron pore size, polysulfone membrane. The genetically engineered cells cannot escape from the bandage, but the bGH is freely released into the surrounding culture medium. When placed onto a full-thickness, surgically generated wound on rats, the cells within the bandage continue to produce and release bGH into the wound for at least 3 days. This system is a safe and reliable way of providing real-time delivery of any desired biomolecule into the wound site.

Cardiovascular actions of vasopressin at the ventrolateral medulla.

Vasopressin acts at a number of sites in the central nervous system to alter arterial pressure. This study investigated the hypothesis that vasopressin acts at the rostral ventrolateral medulla to increase arterial pressure. The rostral pressor area of the medulla oblongata was exposed in urethane-anesthetized rats prepared for topical application of vasopressin. A 3-minute application of vasopressin (range 10(-8) to 10(-3) M) produced dose-dependent increases in arterial pressure that averaged between 2 +/- 1 and 65 +/- 11 mm Hg (p less than 0.01). Tachycardia was not a consistent response at any concentration of vasopressin. Intravenous administration of a V1 vasopressin antagonist did not modify the pressor response produced by topical application of vasopressin (10(-4) M). Application of the V1 antagonist to the rostral pressor area, however, prevented the production of a pressor effect to subsequent topical application of vasopressin (10(-4) M). These experiments suggest that vasopressin stimulates the activity of vasomotor neurons in the rostral ventrolateral medulla by a mechanism that involves a neuronal V1 receptor.

Cardiorespiratory effects induced by acetazolamide on the ventromedullary surface of the cat.

1. Inhibition of carbonic anhydrase by acetazolamide in alpha-chloralose-anaesthetized cats, in a region of the brain stem co-extensive with the glycine-sensitive area, intermediate chemosensitive area, and probably C1 catecholaminergic neurones produces hypotension, bradycardia and depression of the central respiratory drive. 2. These responses are concentration dependent, and can still be observed when the enzyme substrate (CO2) is elevated. Therefore, in both the hypercapnic and the normocapnic condition, similar responses in arterial blood pressure, heart rate and respiratory rate are observed when acetazolamide is topically applied to the glycine-sensitive area. 3. To investigate further the contribution of peripheral baro-, chemo- and cardiopulmonary receptors to these responses, acetazolamide was topically applied to the glycine-sensitive area under three different conditions: intact gallamine-paralysed (5 mg kg-1 h-1) and artificially ventilated (A), sinoaortic denervated (B), and sinoaortic denervated plus bilaterally vagotomized cats (C). Under all conditions, similar responses were observed. The fall in arterial blood pressure was 75 +/- 11 (A), 90 +/- 13 (B), and 75 +/- 9 mmHg (C). Changes in heart rate during acetazolamide application were -23 +/- 6, -20 +/- 8, and -26 +/- 6 beats min-1, respectively. The decreases in respiratory rate were 9 +/- 2 (A), 11 +/- 2 (B), and 11 +/- 2 breaths min-1 (C). 4. The data indicate that the responses to topical application of acetazolamide are mainly due to its central action at the glycine-sensitive area and are not influenced by peripheral baroreceptor and chemoreceptor inputs.