Microscope Photometry Research Articles

Mechanism of destruction of caking property of a coking coal using low-temperature pyrolysis treatment

A low-temperature pyrolysis treatment (LTPT) to destroy the caking property of a coking coal is proposed. Physicochemical properties of the coal before and after LTPT were investigated using Fourier transform infrared spectroscopy, thermogravimetric analysis, automatic microscope photometry, and gas chromatography with mass spectrometric detection. Hot-stage microscopy was used to show in-situ morphological changes in metaplast formation during the LTPT. The results showed that the caking index (G) of the coking coal decreased with an increase in temperature, reaching zero at 468 °C. The lower the vitrinite content, the lower was the G value of the treated sample. The coal began to produce metaplast at 445 °C, which increased with increasing temperature until it re-solidified at 540 °C. Gas chromatography showed that the tars contained large amounts of long-chain aliphatics with 12–24 carbons and polycyclic aromatics with 2–4 rings. The decrease in G value is related to volatilization of these species. Volatilization of long-chain aliphatics leads to loss of H radicals during pyrolysis. The resulting compounds cannot stabilize relatively large radicals, at the core of which are polycyclic aromatics, to produce metaplast. As a result, these relatively large aromatic radicals cross-link with each other to form semicoke.

Thermal maturity and chemical structure evolution of lump long-flame coal during superheated water vapor–based in situ pyrolysis

Oil and gas products can be successfully obtained from low-rank coal using superheated water vapor in situ pyrolysis technology, and investigating the pyrolysis properties of coal during this process is crucial. Herein, the in situ pyrolysis of lump long flame coal with superheated water vapor using a proprietary superheated water vapor pyrolysis system was simulated. The vitrinite reflectance (Ro) and functional groups of coal samples were analyzed using a microscope photometry and Fourier transform infrared spectroscopy. Moreover, the relationship between the chemical structural parameters and vitrinite reflectance during different thermal maturation processes was examined. The results indicate that the vitrinite reflectance increased from 1.01% for raw coal to 2.2% at 550 °C as the pyrolysis temperature increased. The ranges of the critical temperature for the removal of aliphatic structures, C–O, CO, –OH, and aromatic structures from the coal were 271°C-506 °C, 333°C-550 °C, 220°C-550 °C, RT–333 °C, and 333°C-550 °C, respectively. As Ro increased, the aromaticity and condensation both increased linearly and then decreased; the CH2/CH3 ratio first decreased and then increased; the C-factor exhibited negative exponential reduction. Artificial thermal maturation was more effective than natural thermal maturation in promoting the evolution of the aliphatic structure but less effective in promoting the condensation of aromatic rings. Both types of thermal maturation have the same effect on the evolution of aromaticity and C-factor.

Microscopic photometry and its parameterization for objective evaluation of aesthetics of woven fabrics

One of the most important end-use performance parameters for fabrics and other textile products are the fabric hands and fabric aesthetics. Our previous research attempted to elucidate the factors that affect the fabric aesthetics using factor analysis and identified two principal factors tentatively labelled as ‘lustre and depth sensation’ and ‘surface roughness sensation’. The focus of the second step in the study of fabric aesthetics is on the investigation of the objective trends in the performance evaluation of textiles. In this study, we developed our own experimental equipment based on microscopic goniophotometric principles and its parameterization method. Subsequently, we attempted to define an index based on the distribution of the reflection intensity of woven fabrics and its characterization in order to evaluate the principal factors governing fabric aesthetics. On the basis of the results, we infer that microscopic goniophotometry and its parameterization is a more viable approach than conventional macroscopic goniophotometry for the quantitative evaluation of the principal factors governing fabric aesthetics.

Iridescence in beef caused by multilayer interference from sarcomere discs

Microscope photometry of raw and cooked iliocostalis was used to test the hypothesis that interference colours in beef may originate from reflections from sarcomere discs. Evidence in support of the hypothesis was, firstly, that interference colours were not altered by rotating a polarizer in the illumination pathway, or by rotating a polarizer in the measuring pathway. But when both pathways contained polarizers, iridescence was completely extinguished when the polarizers were crossed. Secondly, the reflectance spectra of interference colours all showed multiple interference peaks, with a major peak possibly originating from the top sarcomere and minor peaks originating from lower sarcomeres. Thirdly, major peaks were strongly dependent on the angle of measurement. Iridescence in beef is quite rare, but reflections from sarcomere discs may be a ubiquitous source of light scattering in meat.

Improved detection of nicotinamide adenine dinucleotide phosphate oscillations within human neutrophils

Kinetic studies of nicotinamide adenine dinucleotide phosphate autofluorescence have been conducted in adherent neutrophils using an improved microscopic photometry system incorporating low noise excitation and detection systems. Dynamic autofluorescence oscillations were found with periods ranging from ∼4 min to ∼10 s. The largest portion of the population of oscillating neutrophils (32%) had periods near 2 min. The next largest group at 25% exhibited periods of 1 min or less. These oscillations could not be accounted for by instrument artifacts, cell shape changes away from the focal plane, or other factors. They disappeared when detergent was added to oscillating cells. Higher-frequency oscillations disappeared as cells changed shape, indicating a correlation between these two processes. This approach provides a reliable method to monitor this cellular property.

High-speed digital imaging method for ciliary beat frequency measurement

The aim of this study was to develop a high-speed digital imaging system and related software for ciliary beat frequency (CFB) analysis in order to establish an automated and reliable method that is observer independent and faster compared to the conventional computerized microscope photometry method. Using primary human nasal epithelial cell cultures, the CBF was recorded with a computerized microscope photometry system and a high-speed digital imaging system. To obtain a wide range of frequencies, glycocholate (0.5%) and chlorocresol (0.005%) were used as ciliostimulatory and cilio-inhibitory reference compounds, respectively. The mean values in hertz (+/- s.d.) obtained with the photometry and high-speed digital imaging systems were: controls 8.2 +/- 0.9 and 7.9 +/- 1.1; chlorocresol 5.0 +/- 0.9 and 5.1 +/- 1.1; glycocholate 9.8 +/- 1.0 and 9.7 +/- 0.8. A similar increase (by 20 and 24%) and decrease (by 38 and 35%) in CBF was determined by the two methods after glycocholate and chlorocresol treatment, respectively. The mean difference between the photometry and high-speed digital imaging methods was 0.2 +/- 0.6 Hz, and the Bland-Altman limits of agreement were from -1.0 to +1.4 Hz, suggesting that the results obtained by these two methods could be used interchangeably. These results show the reliability of the high-speed digital imaging system and the software developed for in-vitro CBF measurements. The advantages of the system include: (i) fast data acquisition and calculation, (ii) whole field automated CBF analysis and (iii) reduction in selection bias.

The Role of Protein Kinase C as a Regulator of NG115‐401L Neuronal Cell Calcium Signaling Pathways

The molecular regulation of non-voltage gated calcium influx continues to be an important but poorly characterized signaling pathway in cellular physiology. We have previously characterized unique features of calcium influx in the NG115-401L (401L) neuronal cell line, revealing a phenotype that appears to depend on a conformational coupling mode for the regulation of both calcium influx and release. In this study, we used ratiometric microscope photometry on Fura 2 loaded 401L cells to further probe the apparatus mediating calcium influx when cells are stimulated with pro-inflammatory hormones such as bradykinin. In particular, we have examined the effects of pharmacological modulation of protein kinase C (PKC) on native 401L cells and on cells overexpressing canonical transient receptor potential (TRPC) ion channels. Our preliminary results reveal that inhibitors of PKC augment calcium influx in 401L cells suggesting that PKC activation serves to suppresses the calcium influx mechanism. In contrast, pharmacological activation of PKC using phorbol ester treatment did not significantly influence calcium influx in both native or TRPC overexpressing 401L cells. Intriguingly, we observed that PKC activation in the presence of extracellular calcium elicited discharge from intracellular calcium stores, suggesting a composite target containing PKC activity regulating both calcium release and influx. This work was supported by a grant from the National Institutes of Neurological Disorders and Stroke (1R15 NS048041-01A1) to D.W.T.

Stable ciliary activity in human nasal epithelial cells grown in a perfusion system

Purpose: Explore the usefulness of a perfusion system in order to establish human nasal epithelial cell cultures suitable for long-term in vitro ciliary beat frequency (CBF) and cilio-toxicity studies. Methods: The cells were obtained by protease digestion of nasal biopsy material. The cells were plated at a density of 0.8–1 × 10 6/cm 2 on Vitrogen-coated polyethylene terephthalate membranes, and cultured under submerged conditions in a CO 2 incubator or in a perfusion system (initiated on days 8–9 after plating). The CBF was determined at 24.1 ± 0.8 °C by a computerized microscope photometry system. The morphology of the cultured cells was characterized by transmission electron microscopy (TEM). Results: Under CO 2 incubator culture conditions, stable ciliary activity was expressed and maintained from day 2 to day 24. Under perfusion system culture conditions, the CBF (mean ± S.D., n = 4) amounted to 8.4 ± 0.9 and 8.8 ± 0.4 Hz on days 7 and 14, respectively. These values were lower as compared to the corresponding CBF obtained in the CO 2 incubator cultures (9.5 ± 0.6 and 9.9 ± 1.0 Hz, respectively). Reference cilio-stimulatory (glycocholate) and cilio-inhibitory (chlorocresol) compounds were used to assess CBF reactivity. In the CO 2 incubator and 7- and 14-days perfusion system cultures, glycocholate (0.5%) showed a reversible cilio-stimulatory effect of 23, 26 and 21%, respectively, while chlorocresol (0.005%) exerted a reversible cilio-inhibitory effect of 36, 40 and 36%, respectively. TEM revealed polarized cuboidal to columnar epithelial morphology, with well-differentiated ciliated cells under CO 2 and perfusion system conditions (up to day 23). Conclusion: Culturing human nasal epithelial cells on Vitrogen-coated polyethylene terephthalate membranes in submerged conditions in a CO 2 incubator and in a perfusion system offers the possibility for long-term preservation (up to 22–24 days) of stable and reactive CBF in vitro.

Flower Color Development and Nano‐Science

AbstractFor Abstract see ChemInform Abstract in Full Text.

花の色とナノサイエンス

Red, purple and blue flower color is mainly due to anthocyanins. The color development and stability of anthocyanin are effected by co-existence of co-pigment and metal ions, and pH. Our chemical studies elucidated that the color is stabilized and varied by formation of a supramolecule. Especially beautiful blue flower colors, found in blue dayflower and blue salvia, are developed by a metalloanthocyanin, a stoichiometric supramolecular metal complex pigment.The metalloanthocyanins are composed with six molecules of anthocyanins, six molecules of glycosylflavones, and 2 atoms of metal ion and the diameter is ca. 3 nano meter. Under formation of metalloanthocyanin fine chiral molecular recognition occurs. To clarify the mechanism we synthesized chiral glycosylflavones derivatives according to our newly invented method of direct C-and O-glycosylation to the flavonoid nucleus. Reproduction experiment from the components (anthocyanin, flavone and metal ion) including chiral isomer of the flavones bearing L-glucose was carried out. As a result, the 4'-D-glucoside of the flavone is essential for formation of metalloanthocyanin, but the L-sugar is not.As the petal tissue is not homogenous, both the epidermides are colored. For elucidation of true flower color development in a living petal cell, various analytical methodologies including nano-technology must be applied. For this purpose one cell analysis by pH-microelectrode, a nano-HPLC, a pico mole metalanalysis and microscopic photometry were carried out. The mech-anism of flower color change of morning glory, Ipomoea tricolor, from purple to blue during blooming, and color variation of hydrangea was studied.

Safety-assessment of 3-methoxyquercetin as an antirhinoviral compound for nasal application: effect on ciliary beat frequency

It has been shown that 5,7,3′,4′-tetrahydroxy-3- O-methylflavone (3-MQ) exhibits antipicornaviral activity. In order to explore the potential of 3-MQ as an antirhinoviral compound for nasal application, the effect of 3-MQ on the ciliary beat frequency (CBF) of human nasal epithelial cells was studied in vitro in the absence or presence of solubility/absorption enhancers (hydroxypropyl-β-cyclodextrin (HP-β-CD) or polysorbate 80). Nasal epithelial cells were obtained by protease digestion of surgical specimens of human nasal polyps, and used at confluency. The effect of 3-MQ (2, 10, and 20 μg/ml), HP-β-CD (1, 3, and 10% (w/v)), polysorbate 80 (0.1, 0.3, and 1% (w/v)), and of the combination of 3-MQ with 3% HP-β-CD or 1% polysorbate 80, on the CBF was determined by computerized microscope photometry 15 min after incubation with the test compounds; recovery was determined 35 min after rinsing. HP-β-CD at 1 and 3% did not affect CBF; a reversible decrease (by 37%) was observed at 10%. Polysorbate 80 caused a reversible cilio-inhibitory effect of 40, 53, and 49% at 0.1, 0.3, and 1%, respectively. At 2 and 10 μg/ml, 3-MQ showed a reversible cilio-stimulatory effect of 18 and 14%, respectively. Combined with 3% HP-β-CD, the reversible cilio-stimulatory effect of 2 μg/ml 3-MQ was preserved, while 10 and 20 μg/ml 3-MQ did not affect the CBF. The combination of polysorbate 80 (1%) and 3-MQ decreased the CBF, which could be attributed to the presence of polysorbate 80. In conclusion, no ciliotoxic effect could be observed for 3-MQ (up to 20 μg/ml) in the absence or presence of HP-β-CD (3%). The potential of this combination as an antirhinoviral formulation for nasal application will be further explored.

Mechanistic appraisal of the effects of some protease inhibitors on ciliary beat frequency in a sequential cell culture system of human nasal epithelium

The aim of this study was to investigate the suitability of a sequential monolayer-suspension culture system as a model to screen subacute effects of drug excipients on ciliary beat frequency (CBF). The CBF of the cultured cells was measured by computerized microscope photometry. Protease inhibitors (puromycin, bestatin, bacitracin, actinonin and thiomersal) were used as model compounds and the mechanisms of ciliary inhibition were investigated by probing the involvement of arachidonic acid metabolism, guanylate cyclase (cGMP), protein kinase C (PKC) and adenosinetriphosphate (ATP) inhibition. Bestatin concentration-dependently reduced CBF by inhibiting arachidonic acid metabolism, cGMP, PKC and endogenous ATP consumption. Thiomersal and DMSO used for dissolving actinonin reduced CBF ( P<0.05) via a non-specific mechanism. Bacitracin (8 mM) and puromycin (135 mM) had no effect on CBF after acute exposure (15–30 min) ( P>0.05), but significantly reduced the CBF by approximately 15.0% following daily15-min exposure for 1 week. This study shows that (i) sequential monolayer-suspension culture system is a valid model to screen both acute and subacute effects of drug excipients on CBF; and (ii) bacitracin, puromycin and actinonin are more cilio-compatible than bestatin and thiomersal and as such are more potentially useful nasal absorption enhancer from ciliotoxicity perspective.

Toxicological investigations of the effects carboxymethylcellulose on ciliary beat frequency of human nasal epithelial cells in primary suspension culture and in vivo on rabbit nasal mucosa

The objective of this study was to investigate the safety of a mucoadhesive carboxymethylcellulose (CMC) formulation for intranasal administration of apomorphine. The effect of different concentrations of CMC on ciliary beat frequency (CBF) was studied using a human nasal epithelial suspension cell culture system. The CBF was determined by computerized microscope photometry. The in vivo rabbit nasal mucosal tolerance of the mucoadhesive polymer was investigated using light microscopy. Twice daily, six rabbits received CMC powder in one nostril and CMC/apomorphine powder in the alternate nostril for 4 weeks. Two control rabbits received air puffs in one nostril and nothing in the alternate nostril. The rabbits were subsequently sacrificed and the stained nasal sections examined microscopically. CMC showed both concentration- and time-dependent inhibitory effects on the CBF. Only mild-to-moderate cilio-inhibition was recorded with the different concentrations of the polymer. CMC (both with and without apomorphine) caused mild-to-moderate inflammation after 4 weeks. Necrosis, squamous metaplasia or ciliary degeneration was not observed. Based on: (1) the mild-to-moderate cilio-inhibition induced by different concentrations of CMC; and (2) the mild-to-moderate nasal mucosal inflammation caused by CMC with and without apomorphine, we conclude that this polymer can be considered as a safe carrier for short-term intranasal administration. However, further investigations are required for its use in the treatment of chronic diseases such as with apomorphine in Parkinson's disease.

Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy.

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

Safety assessment of selected cyclodextrins — effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis

The objective of this study was to assess the cilio-inhibitory effect of a series of cyclodextrins using a human cell suspension culture system exhibiting in vitro ciliogenesis. Enzymatically released human nasal epithelial cells were cultured as sequential monolayer-suspension culture showing in vitro ciliogenesis. Ciliary beat frequency (CBF) was determined by computerized microscope photometry. Among the cyclodextrins investigated (γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, anionic-β-cyclodextrin polymer, dimethyl-β-cyclodextrin and α-cyclodextrin), it was shown that after 30 min of exposure, γ-cyclodextrin (10% w/v), hydroxypropyl-β-cyclodextrin (10.0% w/v) and anionic-β-CD polymer (8.0% w/v) were not significantly cilio-inhibitory ( P>0.05). Similarly, CBF remained stable upon cell exposure to α-cyclodextrin (2.0% w/v) and dimethyl-β-cyclodextrin (1.0% w/v). However, higher concentrations of α-cyclodextrin and dimethyl-β-cyclodextrin resulted in mild to severe cilio-inhibition after 45 min of exposure. The effect of α-cyclodextrin (5.0% w/v; 54±4% cilio-inhibition) was partially reversible while dimethyl-β-cyclodextrin (10% w/v; 36±4% cilio-inhibition) was irreversible. The cilio-inhibition observed in this model was lower than reported for chicken trachea model. Given the fact that (1) irreversible cilio-inhibition observed in this study occurred only at concentrations exceeding those used in pharmaceutical formulations and/or at an unusual exposure time (45 min) and that (2) in an in vivo situation, dilution and mucociliary clearance contribute to further decrease in local concentrations of the applied compound, the results of this study confirm the safety of the cyclodextrins investigated as nasal absorption enhancers.

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

: Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1alpha enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo.

Nitric oxide synthase neurons in different areas of normal aged and Alzheimer's brains

This study investigated the distribution of nitric oxide synthase-containing neurons in the cerebral cortex of individuals with Alzheimer's disease, and compared them with age-matched controls. Paraffin-embedded sections of the frontal (area 10), occipital (area 17) and entorhinal cortices (area 28), and hippocampal formation obtained from 13 autopsy cases were used in the study. Neurons expressing nitric oxide synthase messenger RNA and protein were identified, respectively, by in situ hybridization and immunohistochemistry. Optical densities of nitric oxide synthase-positive neurons were assessed in 50 randomly selected fields of each of the above regions of the cortices, in each case by microscopic photometry. In the frontal cortex of the Alzheimer group, while a decrease in the number of nitric oxide synthase-positive neurons was evident, the nitric oxide synthase neurons, on the other hand, showed an increased optical density in layers II–IV when compared with those of normal ageing. In the occipital cortices, no significant differences in optical density were recorded between the normal ageing and Alzheimer specimens. In the entorhinal cortex, the optical densities of nitric oxide synthase neurons were again similar between the Alzheimer and age-matched control groups. In the hippocampal formation itself, there was an increase of nitric oxide synthase staining in the Alzheimer patients. These results show that (i) nitric oxide synthase neurons are abundant in the human cortex, (ii) the distribution of nitric oxide synthase neurons differs between different cortical regions, and (iii) there are differences between normal ageing and Alzheimer patients in the frontal cortex and the hippocampus.

A flow injection renewable surface technique for cell-based drug discovery functional assays.

A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-à-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.

Nasal ciliary beat frequency is age independent

Evaluate the age-dependency of ciliary beat frequency (CBF) in biopsies after ciliogenesis in culture. Retrospective analysis of CBF and ciliary ultrastructure in biopsies and after ciliogenesis from 203 individuals, aged 3 months to 74 years. All patients with successful culture were included. Ciliogenesis was obtained using the sequential monolayer-suspension culture technique for dissociated nasal epithelial cells. CBF was measured using computerized microscope photometry. Secondary ultrastructural abnormalities were evaluated in transmission electron microscopy. There was no correlation between age and CBF, in either the biopsies (7.0 +/- 2.6 Hz; n = 113) or after ciliogenesis in culture (8.1 +/- 1.3 Hz; n = 203). Even in individuals older than 70 years, CBF was normal in bioptic material (6.7 +/- 1.7 Hz) and after ciliogenesis in culture (7.9 +/- 1.0 Hz). Also, in individuals less than 1 year of age CBF was normal in biopsies as well as after ciliogenesis. CBF correlated inversely with the percentage of secondary ultrastructural abnormalities in the biopsies as seen with transmission electron microscopy: 8.1 +/- 1.8 Hz when ciliary ultrastructure was normal and 3.5 +/- 3.3 Hz in cases of severe secondary ciliary dyskinesia. After ciliogenesis in culture, ciliary ultrastructure was always normal, as was CBF. CBF is age independent but correlates with secondary ultrastructural abnormalities. CBF after ciliogenesis in culture is the intrinsic one.

Multiple G-protein involvement in parathyroid hormone regulation of acid production by osteoclasts

The involvement of multiple G-proteins in parathyroid hormone regulation of acid production was demonstrated in a highly enriched osteoclast population. Osteoclasts were isolated from the endosteum of 2.5 to 3-week-old chicken tibia using sequential enzymatic digestion. Single cell analysis of acid production was accomplished using microscope photometry and vital staining with acridine orange, a hydrogen ion concentration sensitive fluorescent dye. Lithium chloride, an uncoupler of G-proteins from their respective receptors, blocked parathyroid hormone stimulated production of acid. Cholera toxin, which permanently activates Gs-proteins, mimicked PTH stimulation. Pertussis toxin, which prevents receptor interaction with Gi- and Go-proteins, blocked both 10(-8) M and 10(-11) M PTH stimulated acid production, suggesting that the pertussis toxin-sensitive G-protein is utilized at both PTH concentrations. Immunoblots of osteoclast plasma membrane proteins, using a panel of antibodies generated against specific G-protein alpha subunits, revealed a 48 kDa Gs alpha, a 41 kDa Go alpha, a 34 kDa Gi alpha-3, and a unique 68 kDa G alpha subunit, with the 41 kDa and 34 kDa bands being the most intense. Immunoblots of osteoblast plasma membrane proteins had a substantially different profile with the most intense bands being a Gs alpha (48 kDa) and a Go alpha (36 and 38 kDa). The studies suggest the utilization of at least two different G-proteins in the parathyroid hormone regulation of acid formation by osteoclasts, a Gs and a pertussis toxin-sensitive G-protein (Go and/or Gi alpha-3).