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PubMed1986

[Comparative analysis of the drinking behavior of rats after microinjections of angiotensin II and bradykinin into the cerebral ventricles and instillations on the conjunctiva of the eye].

The effects of angiotensin-II and bradykinin intracerebroventricular microinjections and instillations on the conjunctiva on water and alcohol consumption in rats were compared. The identical effects of peptide instillations on the conjunctiva and intracerebroventricular microinjections were demonstrated. Dipsogenic doses of angiotensin-II for instillations on the conjunctiva in intact rats were greater than or equal to 10 micrograms. At the same time the effective peptide doses in drinking rats were similar to intracerebroventricular drug doses.

Alcohol Drinking↗

PubMed1985

Coordination of the contractile activity of bile canaliculi. Evidence from calcium microinjection of triplet hepatocytes.

The discovery that bile canaliculi are capable of spontaneous contractile activity has led to their use in the investigation of the physiology of the liver cell. The contraction of a bile canaliculus is dependent on the network of actin, which is found in the pericanalicular region of the hepatocyte, and agents that inhibit actin filaments interfere with canalicular contraction. Injection of calcium directly into the cytoplasm of one hepatocyte of a cell pair results in contraction. Injection of calcium directly into the cytoplasm of one hepatocyte of a cell pair results in contraction of the canaliculus. To determine whether the contractile activity of adjacent bile canaliculi was coordinated, calcium was injected into one cell of a group of three hepatocytes that formed two neighboring bile canaliculi. Calcium microinjection resulted in contraction of the bile canaliculus contiguous with the microinjected cell and was followed by a contraction of the second, adjacent canaliculus. These secondary contractions occurred after an interval of approximately 45 seconds. The finding that bile canalicular contractions are coordinated with contractions occurring in a sequential fashion supports the hypothesis that, within the liver lobule, transport of bile within the canalicular network results from the coordinated contractions of the canaliculi.

Animals↗

PubMed1985

Microinjection study of renal calcium absorption in the phosphate-depleted rat.

Tracer microinjection studies were performed in chronically phosphate-depleted (CPD) and phosphate-supplemented (PS) rats in order to determine the site of the defect in calcium absorption which developed in CPD. Calcium recovery in the final urine was significantly higher in CPD than in PS rats after microinjections into the early and the late proximal tubules. However, there was no significant difference in the urinary recovery between the two groups after injection into the early and late distal convoluted tubules. The difference in calcium recovery in late proximal studies persisted during PTH infusion. This indicates that there is a defect in calcium absorption in phosphate-depleted rats between late proximal and early distal tubules, which is not correctable with PTH infusion.

Animals↗

PubMed1985

Regulation of catabolism of ribonuclease A microinjected into human fibroblasts.

We are using ribonuclease A (RNase A) as a model protein to study how the degradative rates of proteins are regulated within cells. RNase A and several derivatives can be microinjected into confluent cultures of human fibroblasts using red cell-mediated microinjection. The half-life of RNase A is 80-100 hrs in cells maintained in the presence of serum, and the degradative rate is enhanced approximately two-fold upon serum withdrawal. The ability of fibroblasts to regulate breakdown of this protein depends on a small peptide region within the amino terminal twenty amino acids. This amino terminal peptide from RNase A can be covalently attached to unrelated proteins and will cause their catabolism to become serum responsive. The mechanism of degradation of RNase A involves lysosomal pathways both in the presence and absence of serum, and the enhanced catabolism during serum deprivation results from a two-fold increase in the rate of uptake of the protein by lysosomes. These findings suggest that autophagy, or some other process occuring in serum-deprived cells, can be highly selective.

Fibroblasts↗

PubMed1984

Microinjection of neostigmine into the pontine reticular formation of cats enhances desynchronized sleep signs.

Microinjection of the acetylcholinesterase inhibitor neostigmine into the dorsal pontine tegmentum of intact, freely moving cats produced significant changes in electrographic desynchronized (D) sleep signs and D sleep-like behavior. The percentage, frequency and duration of D sleep signs were increased and the latency to onset of D sleep signs was significantly reduced after neostigmine administration. The effects of neostigmine were dose-dependent and could be blocked by centrally administered atropine. This is the first demonstration that microinjection of an acetylcholinesterase inhibitor into the pons enhances D sleep signs. These data suggest that endogenously released acetylcholine can initiate and maintain the state of D sleep and strongly support the cholinergic hypothesis of D sleep generation.

Acetylcholine↗

PubMed1983

Pressure microinjection of nerve growth factor and anti-nerve growth factor into the nucleus and cytoplasm: lack of effects on neurite outgrowth from pheochromocytoma cells.

Exogenous proteins have been introduced into naive and nerve growth factor (NGF)-primed pheochromocytoma cells by pressure microinjection. Microinjection of cells did affect NGF-induced neurite outgrowth by these cells over periods of up to 3 days. Injected NGF failed to elicit neurite outgrowth or regeneration in the absence of extracellular NGF and failed to enhance it in its presence. Injected anti-NGF IgG failed to inhibit neurite outgrowth or regeneration induced by extracellular NGF. Similar results were obtained for both nuclear and cytoplasmic injections.

Animals↗

PubMed1982

Altered degradation of proteins microinjected into senescent human fibroblasts.

Ribonuclease A was introduced into the cytoplasm of IMR-90 human diploid fibroblasts by red cell-mediated microinjection. Early passage fibroblasts degraded ribonuclease A with a half-life of approximately 90 h in the presence of 10% fetal calf serum and enhanced the degradative rate 1.6-fold upon serum withdrawal. Senescent cells degraded ribonuclease A more slowly with half-lives ranging between 125 and 250 h and had diminished capacities to enhance the catabolism of this protein during serum starvation. Decreased protein degradation in senescent cells was also evident for microinjected RNase S-protein, RNase B, aldolase, lysozyme, and the synthetic copolymer polyglutamate: tyrosine:alanine (1:1:1). These alterations in the mechanisms and regulation of intracellular protein degradation may contribute to several biochemical abnormalities characteristic of aging cells and organisms.

Cell Line↗

PubMed1981

[Use of polyethylene glycol for the microinjection of exogenous protein into cultured murine cells].

Polyethylene glycol was applied to microinject two exogenous proteins: bovine serum albumin and non-histone protein derived from mouse spleen chromatin, into the mouse L-cells. The effectiveness of fusion of mammal (human, in the given case) erythrocytes in which hemoglobin is substituted for the protein under study was shown to be higher than when Sendai virus was used. The microinjected proteins preserve their specificity to subcellular structures.

Animals↗

PubMedDate not supplied

[Microinjection of non-histone chromatin protein derived from mouse spleen into L cells].

Non-histone protein selectively released from mouse spleen nuclei by mild hydrolysis with micrococcal nuclease or DNase I was introduced into L cells by the microinjection method. It is shown that this protein selectively accumulates in cell nuclei. Analysis of the mitotic cells after microinjection of 125I labeled protein demonstrated its location in chromosomes of L cells.

Animals↗

PubMed1994

[Inhibition of gastric myoelectric activity and gastric motility induced by microinjection of substance P into caudate nucleus in mouse].

Changes of gastric myoelectric fast wave, slow wave and gastric motility were studied after microinjection of substance P or ACh into caudate nucleus in an attempt to find the interaction between the two substances. Gastric myoelectric activity and motility were recorded by a RM-6200 four channel recorder and then delivered to a IBM-PC computer for analysis. After microinjection of SP or ACh into caudate nucleus, the gastric myoelectric fast wave and gastric motility were significantly suppressed, while the slow wave was not significantly changed.

Animals↗

PubMed1994

[Microinjection of opioid antagonist into periaqueductal gray blocks formalin-induced alterations of substance P in spinal dorsal horn].

Using formalin injected into the rat's hind paw as nociceptive stimulation, the effect of naloxone and beta-endorphin (beta-EP) antiserum microinjected into the periaqueductal gray (PAG) on the level of substance P like immunoreactivity (SPLI) of the spinal dorsal horn was observed. After formalin injection, a significant increase in SPLI was found as compared with the control (P < 0.01). After naloxone or beta-EP antiserum microinjection, SPLI level in the dorsal horn was decreased (P < 0.05). It is thus suggested that nociceptive stimulation induce the release of beta-EP in the PAG, which, in turn, modulate SP release in the spinal dorsal horn.

Animals↗

PubMed1995

Immunomodulating effects of morphine microinjected into periaqueductal gray.

AIM: To study the effects of morphine on immune system through rat brain periaqueductal gray (PAG). METHODS: Three hours after microinjection of morphine through the implanted steel tubes to PAG, splenic cytokines interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor (TNF), and natural killer cells (NK) activity were measured. RESULTS: Microinjection of morphine (0.5 microL, 3672 ng) into PAG region had no influence on IL-6 and TNF-alpha (production of splenic macrophages, suppressed the natural killer cell (NK) activity and enhanced T-lymphocyte functions, including concanavalin A (Con A)-induced T-cell proliferation, IL-2 and TNF-beta production. Both the suppressive and stimulating actions were blocked by PAG preinjection of the mu opioid receptor antagonist naloxone (0.5 microL, 1 microgram), which alone showed the contrary effect to morphine. CONCLUSION: Morphine affected immunofunctions through opioid receptors in PAG, and the influences on various immunocompetent cells were different.

Animals↗

PubMed1995

Modulation of vestibulospinal reflexes through microinjection of GABAergic agents in the dorsal pontine tegmentum of decerebrate cats.

1. The locus coeruleus (LC) neurons control posture as well as the gain of the vestibulospinal (VS) reflexes either through direct coeruleospinal (CS) projections or by inhibiting the dorsal pontine reticular formation (pRF) and the related medullary inhibitory reticulospinal (RS) systems. After the demonstration that the LC neurons are under the inhibitory control of GABAergic afferents, experiments were performed to investigate whether the same system could also suppress the activity of the dorsal pRF, thus intervening in the control of posture and VS reflexes. 2. In precollicular decerebrate cats, unilateral microinjection into the dorsal pRF of the GABA-A antagonist bicuculline or the GABA-B antagonist faclofen (0.13-0.25 microliter at 2.5 micrograms/microliter saline) decreased the tonic contraction of the ipsilateral triceps brachii, but greatly increased the amplitude of the EMG responses of the same muscle to roll tilt of the animal (at 0.15 Hz, +/- 10 degrees). This increase in gain of the VS reflexes was not associated with great changes in phase angle of the responses, which were always characterized by an increased EMG activity during ipsilateral tilt and a decreased activity during contralateral tilt. The same injection also produced in some instances a slight decrease of the extensor tonus of the contralateral forelimb, associated with a moderate increase in the amplitude of modulation and thus in the response gain of contralateral triceps brachii to labyrinth stimulation. However, when the ipsilateral effects were prominent, the response gain of the contralateral triceps muscle was slightly depressed, while the pattern of response reversed. 3. The changes in posture and reflexes described above appeared 5-10 min after unilateral injection of the GABA antagonists in the pRF, reached the highest values in about 30 min and fully recovered to the control levels within 2 h after the injection. These effects were not due to irritative phenomena following injection of the fluid, since neither changes in posture nor in the response gain of the triceps brachii to labyrinth stimulation were observed after injection of an equal volume of vehicle in the pRF of that side. On the other hand, an increase in postural activity associated with a decrease in the response gain of the ipsilateral and contralateral triceps brachii to animal tilt was observed after unilateral microinjection into the dorsal pRF of GABA agonists. 4. Histological controls indicated that the structure responsible for the postural and reflex changes was located in the dorsal pontine reticular formation, ventral to the LC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

Depression of the vestibulospinal reflex by intravermal microinjection of GABAA and GABAB agonists in the decerebrate cat.

Experiments performed in decerebrate cats have shown that unilateral microinjection into the cerebellar anterior vermis of a GABAA (muscimol) or a GABAB agonist (baclofen) decreased the gain of the vestibulospinal reflex involving the ipsilateral triceps brachii (iVSR). On the contrary, the phase angle of the reflex was not significantly modified. These effects started 5 to 10 min after the injection and persisted for at least 1 to 2 h before disappearing. Just the opposite changes in gain of the VSR were obtained after local microinjection of the GABAA (bicuculline) or the GABAB antagonist (saclofen). The area on which the GABAergic agents were effective was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.0 to 1.4 mm with respect to the midline. This vermal region corresponded to the zone B of the cerebellar cortex, which receives a labyrinth input and projects to the ipsilateral lateral vestibular nucleus, where it exerts a prominent inhibitory influence. It is suggested that GABA agonists inhibit the Purkinje (P)-cells' activity, thus reducing the labyrinthine-induced modulation of the firing rate of these neurons by mossy and climbing fiber afferents. Since the vermal P-cells discharge outphase with respect to the excitatory VS neurons, we may explain why a reduced output of these P-cells results in a reduced gain of the VSR. These experiments provide evidence that the cerebellar anterior vermis exerts a positive influence on the basic VSR gain.

Animals↗

PubMed1994

Attenuation of agonist-induced desensitization of the rat substance P receptor by microinjection of inositol pentakis-and hexakisphosphates in Xenopus laevis oocytes.

Recently, inositol hexakisphosphate (phytic acid) was shown to bind to photoreceptor arrestin and block its interaction with rhodopsin. Such an interaction might predict that inositol polyphosphates could alter G protein-coupled receptor desensitization. To investigate the possible roles of higher inositol polyphosphates on receptor desensitization, we have expressed the rat substance P receptor in Xenopus laevis oocytes. The functional expression of substance P receptor was monitored by voltage-clamp recording of substance P-induced Ca(2+)-dependent Cl- currents. When control oocytes were stimulated with substance P (30 nM), after 10 min of washing the second responses to substance P were approximately 15% of the first responses. Cytosolic injection of inositol pentakisphosphate (100 microM) or inositol hexakisphosphate (100 microM) inhibited the reduction of the second substance P-induced current responses, maintaining the second responses to 57-58% of the initial responses. The protective effects of inositol pentakisphosphate and inositol hexakisphosphate against agonist-induced desensitization were concentration and time dependent and structurally specific, in that inositol hexasulfate and inositol tris- and tetrakisphosphate isomers were inactive. Microinjection of inositol hexakisphosphate did not (a) change the potency of substance P or the sensitivity of the expressed substance P receptor to substance P, (b) inhibit 12-O-tetradecanoylphorbol-13-acetate-induced loss of substance P-induced current responses, or (c) alter the currents elicited by microinjection of inositol-1,4,5-trisphosphate. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate have specific inhibitory effects on the agonist-induced loss of responsiveness of the rat substance P receptor. Moreover, these protective effects of inositol hexakisphosphate against desensitization were also observed with the endogenous lysophosphatidic acid/phosphatidic acid receptor, indicating that this mechanism is not specific to ectopic receptors. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate may be novel pharmacological tools for the study of agonist-induced desensitization.

Animals↗

PubMed1994

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.

Amino Acid Sequence↗

PubMed1977

Behavioural and electroencephalographic effects of intracerebral microinjections of sodium pentobarbital.

The effects of intracerebral microinjections of Nembutal into different brain areas, with the purpose of reproducing the behavioural and EEG signs of barbiturate narcosis, have been studied and analyzed. Cats were implanted for polygraphic study of the sleep-wakefulness cycle and bilateral brain canulae were also placed in strategic brain areas. The most striking results was the controlateral turning after unilateral injections of Nembutal in the nigra, suggesting a loss of balance among the actions of the nigro-striatal-cortical pathways on both sides. The sequence of events produced by intravestibular injections of Nembutal suggests that the drug, when injected intraperitoneally, acts also upon the vestibular system. This data stresses the importance of localized intracerebral microinjections of drugs to determine and differentiate their action mechanisms when injected in an intraperitoneal or intravenous way.

Animals↗

PubMed1996

Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon.

Two classes of affinity-purified polyclonal antibodies directed against the four different domains of gizzard caldesmon were prepared and their specificity was verified by immunochemical assays. One set of antibodies recognized exclusively domain-3 spanning residues 483-578 and the other one included IgG reactive toward the remaining domains-1, -2 and -4 corresponding to residues 1-482 and 581-756. Microinjection into cultured fibroblasts of each antibody preparation was employed to probe the functional importance of the interaction between caldesmon and tropomyosin within living nonmuscle cells. Low concentrations of the antibody to domain-3 caused a rapid, severe and reversible disassembly of the microfilament network. In contrast, the antibodies to domains-1, -2 and -4 were ineffective. The effects of the anti-domain-3 IgG were observed not only with the purified antibody but also when present in the whole caldesmon antiserum serving for its isolation. The microfilament disintegrating activity of this antibody was completely abolished upon preincubation with native caldesmon or a proteolytic caldesmon fragment encompassing amino acids 483-578. In enzyme-linked immunosorbent assay competition experiments, tropomyosin, but not F-actin, significantly decreased the binding of the domain-3 antibody to caldesmon. Consistent with recent mutational studies pinpointing to the contribution of domain-3 in the in vitro binding of cellular caldesmon to tropomyosin, the findings suggest that the microinjected domain-3 antibody selectively disrupts the association of tropomyosin with caldesmon domain-3, thereby destabilizing the protein complex and alleviating its known protective action against F-actin severing in the cell. Thus, our data further highlight the in vivo involvement of this particular domain in the attachment of non-muscle caldesmon to tropomyosin as well as the direct participation of the caldesmon-tropomyosin complex in the cytoskeletal organization of the microfilaments.

Actin Cytoskeleton↗