Ciliary Contraction Research Articles

Effect of cAMP- and cGMP-related drugs on porcine cultured ciliary muscle.

We studied in this work the compounds which affect the contraction of ciliary muscle and are potential ocular hypotensive drugs, and second messengers produced by the compounds. Using second-passaged porcine cultured ciliary muscle, single cell contraction was used for assay. Cell contraction by carbachol was affected by isobutyl methylxanthine, forskolin, dibutyryl cGMP and sodium nitroprusside (SNP). Forskolin increased intracellular cAMP while SNP stimulated cGMP formation. The results indicate that these cAMP- or cGMP-related compounds could affect the contraction of ciliary muscle like prostaglandins and could therefore have a potential to reduce intraocular pressure.

M 3-Type Muscarinic Receptors Predominantly Mediate Neurogenic Quick Contraction of Bovine Ciliary Muscle

1. The present experiments were designed to investigate which subtypes of muscarinic receptors are involved in the neurogenic quick contraction of bovine ciliary muscle in connection to quick eye focal accommodation. 2. Transmural electrical stimulation (TES) produced a transient contraction, which was abolished in the presence of 3×10 −7 M tetrodotoxin and 10 −6 M atropine, but greatly augmented by 3×10 −7 M physostigmine. 3. The exogenously applied acetylcholine (ACh: 10 −9 to 3×10 −6 M) produced a concentration-dependent contraction, which was competitively antagonized by 10 −6 M atropine and augmented by 3×10 −7 M physostigmine, but unaffected by 3×10 −7 M tetrodotoxin. 4. The magnitude and time to peak of the maximal contraction produced by TES were significantly greater (1267.5±86.0 mg, P<0.005) and shorter (9.0±0.2 sec, P<0.005) than corresponding values (97.0±9.9 mg and 20.3±2.1 sec, respectively) of the phasic contraction caused by exogenously applied 10 −5 M ACh, at which concentration the agonist caused the maximal contraction. The velocity (140.6±7.8 mg/sec) of the transient contraction caused by TES was approximately 28-fold greater than that of the phasic contraction caused by ACh (5.1±0.9 mg/sec). 5. The contractions produced by TES were greatly attenuated by 4-diphenylacetoxy- N-methylpiperidine (4-DAMP) as an M 3 antagonist and slightly by pirenzepine as an M 1 antagonist (20.2±7.9% inhibition at the highest concentration), but not by methoctramine (MET) as an M 2 antagonist. The IC 50 value (−log M) for 4-DAMP was determined to be 7.17±0.14. 6. Scatchard plot analysis of [ 3H]-quinuclidinylbenzilate (QNB) binding revealed that the binding sites constituted a single population with a K d of 31.2±0.8 pM and a B max of 895.5±93.2 fmol/mg protein. The activity in inhibiting [ 3H]-QNB binding was most potent with 4-DAMP (-log K i=7.98±0.02), but less potent with pirenzepine (−log K i=6.43±0.04) and MET (−log K i=7.32±0.16). 4-DAMP was approximately 35- and 5-fold more potent than pirenzepine and MET in terms of −log K i values, respectively, suggesting the predominant localization of M 3 receptor subtypes in the bovine ciliary muscle membrane. 7. These results suggest that TES produces a neurogenic quick contraction of the bovine ciliary muscle, which would be mediated mainly by ACh released from the intramural nerve terminals and subsequent excitation of M 3 receptor subtypes localized on the ciliary muscle cells, and that neurogenic quick contraction of the ciliary muscle is possibly involved in part in eye focal accommodation.

Classification of human scleral spur cells in monolayer culture

Aqueous humor outflow in primate eyes can be facilitated by ciliary muscle contraction, thereby widening fluid pathways through the trabecular meshwork. Recently in the scleral spur smooth muscle (sm) alpha-actin positive myofibroblast-like cells have been described which are in contact with the elastic fiber system of both the spur and trabecular meshwork. In the vicinity of these cells nerve terminals have been described. It is speculated that contraction of scleral spur cells can facilitate aqueous humor outflow, too. To provide a tool for further physiological and pharmacological studies monolayer cell cultures of human scleral spur have been established and characterized. For this purpose, cells derived from scleral spur, outer and inner trabecular meshwork and ciliary muscle tips from 7 donor eyes (43-87 years-old respectively, obtained 3-7 h post mortem) were grown in tissue culture medium and the different monolayer cells classified by their growth characteristics, and by immunohistochemical staining for vimentin, alpha-sm-actin, desmin, and alpha B-crystallin, respectively. In addition, the presence of alpha B-crystallin mRNA and desmin mRNA was verified using the polymerase chain reaction (PCR)-method. We were able to characterize and distinguish human scleral spur cells from adjacent ciliary muscle and trabecular meshwork cells. Scleral spur cells (SPC) grew slower than ciliary muscle cells (CMC) but much faster than trabecular meshwork cells (TMC). All cells showed the same staining characteristics in vitro as they did in vivo. Scleral spur cells stained for vimentin and alpha-sm-actin, but not for desmin and alpha B-crystallin. The corresponding mRNAs of the latter two proteins could not be detected by PCR in the spur cells. Cells grew out from all donor eyes so that they actually provide a tool for further experimental studies.

Contraction of Human Ciliary Muscles of Aged Phakic and Aphakic Eys in Response to Carbachol

Contraction of Human Ciliary Muscles of Aged Phakic and Aphakic Eys in Response to Carbachol

P 328 Contraction of human ciliary muscles of aged phakic and aphakic eys in response to carbachol

P 328 Contraction of human ciliary muscles of aged phakic and aphakic eys in response to carbachol

Accommodation and presbyopia

The mechanism of accommodation has been studied for at least four hundred years. The most interesting aspect of accommodation is that its time course is well in advance of other physiological functions —it begins to decline by adolescence and is lost about two-thirds of the way through the normal life span. The state of presbyopia is reached when accommodation has declined sufficiently to interfere with close tasks requiring acute vision. Presbyopia is generally considered to originate with the ‘plant’ of the accommodative system, either within the lens and its capsule or within their support structures. One of the lenticular theories, the Hess-Gullstrand theory, is distinguished from other theories by its claim that as age increases there is an increasing excess amount of ciliary muscle contraction beyond the ability of the lens and capsule to respond to it. For all other theories, the maximum possible amount of ciliary muscle contraction is always necessary to produce maximum accommodation, at least beyond the age at which it reaches its peak. From my review of the present understanding of the mechanisms of accommodation and the theories of the development of presbyopia, I conclude that there is overwhelming evidence against the Hess-Gullstrand theory and that it is unlikely that changes in the ciliary muscle contractility contribute significantly to the development of presbyopia.

Accommodation and presbyopia

SummaryThe mechanism of accommodation has been studied for at least four hundred years. The most interesting aspect of accommodation is that its time course is well in advance of other physiological functions – it begins to decline by adolescence and is lost about two‐thirds of the way through the normal line span. The state of presbyopia is reached when accommodation has declined sufficiently to interfere with close tasks requiring acute vision. Presbyopia is generally considered to originate with the ‘plant’ of the accommodative system, either within the lens and its capsule or within their support structures. One of the lenticular theories, the Hess–Gullstrand theory, is distinguished from other theories by its claim that as age increases there is an increasing excess amount of ciliary muscle contraction beyond the ability of the lens and capsule to respond to it. For all other theories, the maximum possible amount of ciliary muscle contraction is always necessary to produce maximum accommodation, at least beyond the age at which it reaches its peak. From my review of the present understanding of the mechanisms of accommodation and the theories of the development of presbyopia, I conclude that there is overwhelming evidence against the Hess–Gullstrand theory and that it is unlikely that changes in the ciliary muscle contractility contribute significantly to the development of presbyopia.

The Effect of Muscarinic Agonists and Selective Receptor Subtype Antagonists on the Contractile Response of the Isolated Rhesus Monkey Ciliary Muscle

There are ultrastructural and histochemical differences between the longitudinal (putatively more relevant to outflow facility) and circular (putatively more relevant to accommodation) portions of the primate ciliary muscle. Oxotremorine, a muscarinic agonist putatively somewhat selective for the M 2 receptor subtype, binds preferentially to the longitudinal rather than the circular portion. Aceclidine, a putatively non-subtype selective muscarinic agonist, can dissociate accommodative and outflow facility responses in monkeys and humans. We used the muscarinic receptor subtype antagonists 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), 11-{[2-(diethylamino)methyl]-1-piperidinyl}acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116), and piranzepine to inhibit contractile responses to the muscarinic agonists carbachol, aceclidine and oxotremorine in the longitudinal and circular vectors of the rhesus monkey ciliary muscle in vitro. Oxotremorine generated dose-response curves that were similar in both the circular and longitudinal vectors and intermediate to those previously reported for carbachol and aceclidine. 4-DAMP (M 3 selective) was the most potent inhibitor of contractile responses to all three agonists, with IC 50 values ranging from 33 to 68 nM for the circular and from 27 to 63 nM for the longitudinal vector, depending on the agonist used to elicit contraction. Pirenzepine (M 1 selective) was ≥ 25-fold less potent and AF-DX 116 (M 2 selective) was ≥ 108-fold less potent at inhibiting contractile responses to all three agonists in either vector, indicating that M 3 is the predominant receptor subtype mediating ciliary muscle contraction in both vectors. The IC 25, IC 50 and IC 75 values for all three antagonists against aceclidine was approximately 1·4-fold less for the longitudinal than the circular vector, of uncertain relevance to aceclidine's in vivo facility-accommodation dissociation.

Pilocarpine-induced lysosomal enzyme secretion, from bovine trabecular meshwork cells

It has long been held that pilocarpine-induced ciliary muscle contraction causes a mechanical change in the configuration of the trabecular meshwork, thereby increasing its fluid conductance. Though the results of many studies are consistent with this theory, other findings suggest a direct action of pilocarpine on the meshwork. We used cultured bovine trabecular meshwork (BTM) cells to study the cell biological effects of pilocarpine, chemically a weak base, and demonstrated increased vacuolization of the BTM cells after incubation with pilocarpine, reflecting a trapping of the protonated base inside the lysosomes. We also showed that pilocarpine enhanced the release of lysosomal hydrolases into the medium. We hypothesize that this is clinically relevant and that the pilocarpine-induced release of hydrolases modifies the extracellular matrix of the trabecular meshwork, thereby increasing its fluid conductance.

Age does not affect contractile responses of the isolated rhesus monkey ciliary muscle to muscarinic agonists.

In primates, ciliary muscle contraction causes accommodation and facilitates aqueous outflow. In living rhesus monkeys, accommodative, outflow facility, and ciliary muscle movement responses to cholinergic agonists all decline with age. We developed an apparatus to determine in vitro whether the latter is related to intra- or extra-ciliary muscle factors, and whether ciliary muscle contraction in the coronal (putatively more accommodation-relevant) and longitudinal (putatively more facility-relevant) vectors can be dissociated pharmacologically. In fresh ciliary muscle strips, carbachol and aceclidine each induced dose-dependent contraction in the longitudinal and coronal vectors. With neither drug was there any apparent dissociation of the responses in the two vectors. Atropine pretreatment completely prevented a supramaximal dose of carbachol from inducing ciliary muscle contraction in either vector. Ciliary muscle strips responded to several cholinergic agonists as well on day 2 (24-32 hours post-enucleation) as on day 1 (1-9 hours post-enucleation) when kept in a cell culture medium at 4 degrees C. By light microscopy, the general architecture of the ciliary muscle, the muscle bundles, and the single muscle cells appeared normal; however, cellular and nuclear swelling were apparent following the 32-hour culturing period. Contractile responses to near-maximal doses of carbachol and aceclidine did not vary markedly with age in either vector, suggesting that the age-related decrease in ciliary muscle mobility in vivo is due to extra-muscular restrictive factors rather than diminished muscular contractility.

Source of aqueous humor hydrogen peroxide

Purpose: To quantify intraocular inflammation after phacoemulsification with implantation of an accommodative posterior chamber intraocular lens (IOL).Setting: Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany.Methods: Twenty cataractous eyes of 20 patients without preexisting blood-aqueous barrier (BAB) deficiencies or previous intraocular surgery were included in this study. The mean age of the patients was 64.6 years ± 16.0 (SD). A single surgeon performed phacoemulsification through a superior sclerocorneal tunnel incision and implantation of a 1CU IOL (HumanOptics AG) though a 3.2 mm incision. The haptics of the single-piece acrylic 1CU lens are designed for anterior optic movement following ciliary muscle contraction. The postoperative treatment was standardized. Postoperative BAB breakdown was quantified by laser flare photometry (FC-1000, Kowa) at 1 day, 1 and 4 weeks, and 3 and 6 months.Results: The mean aqueous flare was 6.3 photons/ms ± 3.0 (SD) (range 4.0 to 12.2 photons/ms) 1 day postoperatively, with 64% of patients having normal aqueous flare values (<8.0 photons/ms). One week after surgery, the mean aqueous flare was 5.3 ± 2.8 photons/ms (range 2.0 to 10.5 photons/ms). Four weeks postoperatively, aqueous flare was normal in all patients and remained stable below the normal limit for up to 6 months (mean 3.3 ± 1.2 months; range 2.0 to 5.4 months). The number of aqueous cells did not increase at any follow-up and was normal in all eyes. No postoperative complications such as fibrin formation, synechias, macrophages on the IOL optic, or endophthalmitis were observed.Conclusions: Phacoemulsification with implantation of the 1CU accommodative IOL led to minimal and short-lasting BAB alteration. No signs of persistent inflammation or pigment dispersion were detected.

Age-related changes of the human ciliary muscle. A quantitative morphometric study

The age-related changes of the ciliary muscle of human eyes (33–87 years) were studied on histological meridional sections. Eighty-five melanoma eyes and 10 eyes of normal donors were investigated. The total area and the length of the muscle, the area of the three main portions and the distance of the inner apex of the muscle to the scleral spur were determined and correlated with age. Total area and length of the muscle show a continuous and significant decrease with age. The area of the longitudinal and reticular portion continuously decreases, whereas the area of the circular portion significantly increases with age. The decrease in area is more pronounced in the longitudinal portion than in the reticular portion of the muscle, which shows an age-related increase in connective tissue. In addition, the distance of the inner apex of the muscle to the scleral spur shortens continuously. Thus, with increasing age the ciliary muscle adopts an anterior-inward position. A similar form is seen in young eyes after ciliary muscle contraction only. There might be a functional relationship between the observed age-changes in the ciliary muscle system and the phenomenon of the so-called ‘lens paradox’ (steepening of the anterior and posterior curvatures of the disaccomodated lens with age).

Effect of phosphodiesterase inhibitors on bovine ciliary muscle and outflow facility.

To understand the aqueous outflow mechanism, we compared changes in the outflow facility with the response of ciliary muscle in fresh bovine eyes. The facility was measured in intact ocular globes. Theophylline, caffeine, and isobutylmethylxanthine (IBMX), all phosphodiesterase (PDE) inhibitors, increased outflow facility in a dose-dependent manner. However, neither epinephrine nor isoproterenol increased the outflow facility, regardless of their concentrations up to 10(-3) M. Neither epinephrine nor isoproterenol (0.01-1 microM)) relaxed the tone of bovine ciliary muscle, whereas theophylline and IBMX remarkably relaxed the muscle which had been contracted by carbachol, and also inhibited the nerve-mediated contraction. It is interesting that PDE inhibitors have a much greater influence on changes in outflow facility and ciliary muscle contraction than beta-adrenergic agonists.

Measurements of intracellular calcium and contractility in human ciliary muscle

Electromechanical and pharmacomechanical coupling was investigated in human ciliary muscle by measuring the intracellular free calcium in single cultured ciliary muscle cells and the contractility in meridional ciliary muscle strips. The basal resting calcium concentration was 75 +/- 8.7 nmol/l, n = 23. Application of acetylcholine (0.1 mmol/l) and carbachol (0.1 mmol/l) resulted in an initial [Ca2+]i peak followed by a recovery phase and a [Ca2+]i plateau. The initial [Ca2+]i peak was still observed in the absence of extracellular calcium and in the presence of verapamil (0.1 mmol/l). During its plateau [Ca2+]i was decreased by withdrawal of extracellular calcium or application of verapamil (0.1 mmol/l). Depolarization induced by a high level of extracellular potassium yielded only a small transient [Ca2+]i peak without a [Ca2+]i plateau. In isolated ciliary muscle strips, muscarinic stimulation (carbachol 0.1 mmol/l) resulted in an initial phasic and a subsequent tonic contraction. Removal of external calcium reduced the phasic contraction to 30.6 +/- 4.4% (n = 8) and completely abolished the tonic one. Verapamil (0.1 mmol/l) had only a slight relaxing effect when applied during the tonic contraction. We conclude that human ciliary muscle contraction is mediated by calcium release from intracellular stores and calcium entry through calcium channels, which are most probably receptor-operated. Depolarization of the muscle cell membrane and calcium entry through voltage-operated calcium channels do not contribute significantly to human ciliary muscle contraction.

Contraction of porcine ocular ciliary muscle by epidermal growth factor-urogastrone

Epidermal growth factor-urogastrone (EGF) caused a concentration-dependent contractile response in porcine ocular ciliary muscle preparations. The half-maximal contraction was observed at 23 ng/ml EGF (4 nM). The contractile action of EGF, which was not abolished by the removal of extracellular calcium, was not affected by atropine, tetrodotoxin, phentolamine and indomethacin. In addition to causing contractions on its own, the contractile action of EGF was potentiated in the presence of prostaglandin E 1 and vasopressin. Our data point to a potential role for EGF in the regulation of ciliary muscle tension.

Reproducible stimulation of ciliary muscle contraction in the cynomolgus monkey via a permanent indwelling midbrain electrode

To better study the pivotal role of the ciliary muscle in ocular accomodation and aqueous humor outflow, a technique was developed to permit long-term reproducible stimulation of the muscle via the normal efferent neuronal pathway in cynomolgus monkeys. Following a small midline craniotomy, the stereotaxic coordinates of the Edinger-Westphal (E-W) nucleus were localized by contrast ventricularadiography. A bipolar stimulating electrode was stereotaxically positioned to generate the maximum accommodative amplitude, and then permanently affixed to the surrounding cranium. Once experience was gained, the implant procedure required ∼3 h and was well-tolerated by the animals, which subsequently exhibited normal behavior and no apparent discomfort or neurological deficits. Most animals retained the implant seemingly permanently (8–42 months follow-up), although a few animals extruded them after 4–8 months. Electrode placement in the E-W nucleus was verified histologically. The stimulation current and frequency-accommodative response relationships varied remarkably little between eyes, animals, or experimental sessions, although there was moderate inter-animal variation in the maximum accommodative amplitude. Using square-wave pulses of 0.5 ms duration, maximum accommodation, averaging ∼15 diopters, was achieved at ∼700 μA and ∼70 Hz, and was greater under pentobarbital than under halothane anesthesia. Topical application of carbachol, eserine and echotiiophate to the eye induced more accommodation than did central electrical stimulation. Permanent implantation of a stimulating electrode into the E-W nucleus may facilitate study of ocular physiological parameters dependent upon ciliary muscle contraction.

Adenosine 3′,5′-cyclic-monophosphate and outflow facility in monkey eyes with intact and retrodisplaced ciliary muscle

Bolus intracameral infusion of 100 micrograms cAMP, corresponding to an initial intracameral concentration of 3 mM, significantly increased total outflow facility in cynomolgus monkey eyes with intact or disinserted-retrodisplaced ciliary muscles by 41% and 37% respectively. Lower cAMP doses had little or no effect in both types of eyes, as did dibutyryl cAMP doses of 1-100 micrograms (20 microM-2mM). In eyes with intact ciliary muscles, intracameral infusion (1 and 10 micrograms) of the cAMP analog 8-methylthio cAMP (0.25 mM and 2.5 mM) significantly increased facility by 55% and 69% respectively, while 100 micrograms of the inactive cAMP metabolite 5'AMP (3 mM) did not alter facility. The facility increases produced by cAMP and 8-methylthio cAMP represented a constant percentage of starting facility independent of the absolute value of starting facility. These findings indicate that the facility increase produced by cAMP and appropriate analogs is not mediated by ciliary-muscle contraction and that the disinsertion procedure itself does not compromise the ability of the outflow pathways to respond to cAMP. The facility increase probably represents a cAMP effect directly on the endothelial cells of the trabecular meshwork-Schlemm's canal inner wall.

Review of ciliary muscle effort in presbyopia.

Fincham's theory of presbyopia would require maximum ciliary muscle contraction to produce maximum accommodative response at all ages. The Hess-Gullstrand theory, however, would allow a maximum accommodative response with a decreasing ciliary muscle contraction as age increases leaving a reserve ability of ciliary muscle to contract with age. If Fincham's theory is correct, a mild parasympatholytic drug would produce a decrease in the amplitude of accommodation, a mild parasympathomimetic drug would produce an increase in the amplitude of accommodation, and the AC/A ratio would increase with age. The results of this study indicate that all three of these do occur and therefore support the Fincham concept of the cause of presbyopia.

Neuronal influence on the mechanical activity of the ciliary muscle.

1 Neuronal effects and the pharmacological properties of the bovine ciliary muscle were investigated in vitro. The bovine ciliary muscle exhibited no spontaneous activity. 2 Electrical stimulation of an isolated short ciliary nerve produced distinct contractions. The minimal stimulus duration required to evoke a contraction was 0.2 ms and amplitude of the contraction was maximal at 2 ms. Twitch or incomplete tetanus reached a complete tetanus with 4 Hz stimulation. 3 Raising the external potassium concentration from 5.9 to 158.8 mM produced a contracture which consisted of an initial phasic and then tonic components. 4 The contractions generated by either electrical stimulation (0.2-100 ms) or high K were potentiated by physostigmine and completely inhibited by atropine. Neither adrenoceptor agonists nor blockers influenced these contractions. 5 Application of tetraethylammonium (TEA), potentiated the electrically-induced ciliary muscle contraction, and the effect of TEA was not completely inhibited by high concentrations of either atropine or tetrodotoxin. Thus, TEA presumably acts both pre-junctionally and post-junctionally to increase the contractile development of ciliary muscle. 6 The ciliary contractile response is primarily mediated by acetylcholine released from nerves, and this response is accompanied by a negligible contribution from the sympathetic nerves. Depolarization induced by electrical currents or by high K was ineffective in evoking contraction of the ciliary muscle. 7 The results suggest that excitation of the ciliary muscle is probably mediated via junction potentials or by a direct transmitter action without any very great change in the potential. Action potentials are probably generated in the presence of TEA.

From the ameba to the pulsating heart: evolution and fine structure of the intracellular movement apparatus (author's transl)

Different kinds of cell motility are reviewed in this paper with special regard to development and ultrastructure. The variety of animal cell motility types can be reduced to three principles : ciliary and ameboid movements and muscle contraction. The ultrastructure of all kinds of cilia is very similar from single cell organisms to highly specialized cells of the human body e.g., ciliary respiratory epithelium. As a rule, ciliary movement is caused by minimal sliding of the nine double tubules consisting of tubulin, a protein differing from myosin and actin. Ameboid movement and muscle cell contraction are based on the sliding filament mechanism of actin and myosin. Although the principles of this mechanism have not changed during evolution some differences in the structure and arrangement of actin and myosin filaments occurred. Obviously, the high degree of order of the myofibrils of vertebrate heart and skeletal muscle cells has developed from loose and rapid changing arrangement of contractile filaments in ameboid cells. There are some changes of residues in the actin and myosin molecules during the development of the intracellular contractile system. Finally, some peculiarities of the myocardium, its special arrangement of muscle cells and some disturbances of the contractile filaments under pathologic conditions are discussed.